Karyotype versus microarray testing for genetic abnormalities after stillbirth.

PubMed

Reddy, Uma M; Page, Grier P; Saade, George R; Silver, Robert M; Thorsten, Vanessa R; Parker, Corette B; Pinar, Halit; Willinger, Marian; Stoll, Barbara J; Heim-Hall, Josefine; Varner, Michael W; Goldenberg, Robert L; Bukowski, Radek; Wapner, Ronald J; Drews-Botsch, Carolyn D; O'Brien, Barbara M; Dudley, Donald J; Levy, Brynn

2012-12-06

Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

PubMed Central

Zlobec, Inti; Suter, Guido; Perren, Aurel; Lugli, Alessandro

2014-01-01

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research. PMID:25285857

The CD117 immunohistochemistry tissue microarray survey for quality assurance and interlaboratory comparison: a College of American Pathologists Cell Markers Committee Study.

PubMed

Dorfman, David M; Bui, Marilyn M; Tubbs, Raymond R; Hsi, Eric D; Fitzgibbons, Patrick L; Linden, Michael D; Rickert, Robert R; Roche, Patrick C

2006-06-01

We have developed tissue microarray-based surveys to allow laboratories to compare their performance in staining predictive immunohistochemical markers, including proto-oncogene CD117 (c-kit), which is characteristically expressed in gastrointestinal stromal tumors (GISTs). GISTs exhibit activating mutations in the c-kit proto-oncogene, which render them amenable to treatment with imatinib mesylate. Consequently, correct identification of c-Kit expression is important for the diagnosis and treatment of GISTs. To analyze CD117 immunohistochemical staining performance by a large number of clinical laboratories. A mechanical device was used to construct tissue microarrays consisting of 3 x 1-mm cores of 10 tumor samples, which can be used to generate hundreds of tissue sections from the arrayed cases, suitable for large-scale interlaboratory comparison of immunohistochemical staining. An initial survey of 63 laboratories and a second survey of 90 laboratories, performed in 2004 and 2005, exhibited >81% concordance for 7 of 10 cores, including all 4 GIST cases, which were immunoreactive for CD117 with >95% staining concordance. Three of the cores achieved less than 81% concordance of results, possibly due to the presence of foci of necrosis in one core and CD117-positive mast cells in 2 cores of CD117-negative neoplasms. There was good performance among a large number of laboratories performing CD117 immunohistochemical staining, with consistently higher concordance of results for CD117-positive GIST cases than for nonimmunoreactive cases. Tissue microarrays for CD117 and other predictive markers should be useful for interlaboratory comparisons, quality assurance, and education of participants regarding staining nuances such as the expression of CKIT by nonneoplastic mast cells.

Use of lectin microarray to differentiate gastric cancer from gastric ulcer

PubMed Central

Huang, Wei-Li; Li, Yang-Guang; Lv, Yong-Chen; Guan, Xiao-Hui; Ji, Hui-Fan; Chi, Bao-Rong

2014-01-01

AIM: To investigate the feasibility of lectin microarray for differentiating gastric cancer from gastric ulcer. METHODS: Twenty cases of human gastric cancer tissue and 20 cases of human gastric ulcer tissue were collected and processed. Protein was extracted from the frozen tissues and stored. The lectins were dissolved in buffer, and the sugar-binding specificities of lectins and the layout of the lectin microarray were summarized. The median of the effective data points for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Formalin-fixed paraffin-embedded gastric cancer tissues and their corresponding gastric ulcer tissues were subjected to Ag retrieval. Biotinylated lectin was used as the primary antibody and HRP-streptavidin as the secondary antibody. The glycopatterns of glycoprotein in gastric cancer and gastric ulcer specimens were determined by lectin microarray, and then validated by lectin histochemistry. Data are presented as mean ± SD for the indicated number of independent experiments. RESULTS: The glycosylation level of gastric cancer was significantly higher than that in ulcer. In gastric cancer, most of the lectin binders showed positive signals and the intensity of the signals was stronger, whereas the opposite was the case for ulcers. Significant differences in the pathological score of the two lectins were apparent between ulcer and gastric cancer tissues using the same lectin. For MPL and VVA, all types of gastric cancer detected showed stronger staining and a higher positive rate in comparison with ulcer, especially in the case of signet ring cell carcinoma and intra-mucosal carcinoma. GalNAc bound to MPL showed a significant increase. A statistically significant association between MPL and gastric cancer was observed. As with MPL, there were significant differences in VVA staining between gastric cancer and ulcer. CONCLUSION: Lectin microarray can differentiate the different glycopatterns in gastric cancer and gastric ulcer, and the lectins MPL and VVA can be used as biomarkers. PMID:24833877

Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

PubMed

Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

2010-01-01

The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

Microarray profiles reveal that circular RNA hsa_circ_0007385 functions as an oncogene in non-small cell lung cancer tumorigenesis.

PubMed

Jiang, Ming-Ming; Mai, Zhi-Tao; Wan, Shan-Zhi; Chi, Yu-Min; Zhang, Xin; Sun, Bao-Hua; Di, Qing-Guo

2018-04-01

Circular RNAs (circRNAs) are a novel class of non-protein-coding RNA. Emerging evidence indicates that circRNAs participate in the regulation of many pathophysiological processes. This study aims to explore the expression profiles and pathological effects of circRNAs in non-small cell lung cancer (NSCLC). Human circRNAs microarray analysis was performed to screen the expression profile of circRNAs in NSCLC tissue. Expressions of circRNA and miRNA in NSCLC tissues and cells were quantified by qRTPCR. Functional experiments were performed to investigate the biological functions of circRNA, including CCK-8 assay, colony formation assay, transwell assay and xenograft in vivo assay. Human circRNAs microarray revealed a total 957 abnormally expressed circRNAs (> twofold, P < 0.05) in NSCLC tissue compared with adjacent normal tissue. In further studies, hsa_circ_0007385 was significantly up regulated in NSCLC tissue and cells. In vitro experiments with hsa_circ_0007385 knockdown resulted in significant suppression of the proliferation, migration and invasion of NSCLC cells. In vivo xenograft assay using hsa_circ_0007385 knockdown, significantly reduced tumor growth. Bioinformatics analysis and luciferase reporter assay verified the potential target miR-181, suggesting a possible regulatory pathway for hsa_circ_0007385. In summary, results suggest hsa_circ_0007385 plays a role in NSCLC tumorigenesis, providing a potential therapeutic target for NSCLC.

Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.

PubMed

Johnson, Jason M; Castle, John; Garrett-Engele, Philip; Kan, Zhengyan; Loerch, Patrick M; Armour, Christopher D; Santos, Ralph; Schadt, Eric E; Stoughton, Roland; Shoemaker, Daniel D

2003-12-19

Alternative pre-messenger RNA (pre-mRNA) splicing plays important roles in development, physiology, and disease, and more than half of human genes are alternatively spliced. To understand the biological roles and regulation of alternative splicing across different tissues and stages of development, systematic methods are needed. Here, we demonstrate the use of microarrays to monitor splicing at every exon-exon junction in more than 10,000 multi-exon human genes in 52 tissues and cell lines. These genome-wide data provide experimental evidence and tissue distributions for thousands of known and novel alternative splicing events. Adding to previous studies, the results indicate that at least 74% of human multi-exon genes are alternatively spliced.

The tissue microarray OWL schema: An open-source tool for sharing tissue microarray data

PubMed Central

Kang, Hyunseok P.; Borromeo, Charles D.; Berman, Jules J.; Becich, Michael J.

2010-01-01

Background: Tissue microarrays (TMAs) are enormously useful tools for translational research, but incompatibilities in database systems between various researchers and institutions prevent the efficient sharing of data that could help realize their full potential. Resource Description Framework (RDF) provides a flexible method to represent knowledge in triples, which take the form Subject-Predicate-Object. All data resources are described using Uniform Resource Identifiers (URIs), which are global in scope. We present an OWL (Web Ontology Language) schema that expands upon the TMA data exchange specification to address this issue and assist in data sharing and integration. Methods: A minimal OWL schema was designed containing only concepts specific to TMA experiments. More general data elements were incorporated from predefined ontologies such as the NCI thesaurus. URIs were assigned using the Linked Data format. Results: We present examples of files utilizing the schema and conversion of XML data (similar to the TMA DES) to OWL. Conclusion: By utilizing predefined ontologies and global unique identifiers, this OWL schema provides a solution to the limitations of XML, which represents concepts defined in a localized setting. This will help increase the utilization of tissue resources, facilitating collaborative translational research efforts. PMID:20805954

Tissue microarray immunohistochemical detection of brachyury is not a prognostic indicator in chordoma.

PubMed

Zhang, Linlin; Guo, Shang; Schwab, Joseph H; Nielsen, G Petur; Choy, Edwin; Ye, Shunan; Zhang, Zhan; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

2013-01-01

Brachyury is a marker for notochord-derived tissues and neoplasms, such as chordoma. However, the prognostic relevance of brachyury expression in chordoma is still unknown. The improvement of tissue microarray technology has provided the opportunity to perform analyses of tumor tissues on a large scale in a uniform and consistent manner. This study was designed with the use of tissue microarray to determine the expression of brachyury. Brachyury expression in chordoma tissues from 78 chordoma patients was analyzed by immunohistochemical staining of tissue microarray. The clinicopathologic parameters, including gender, age, location of tumor and metastatic status were evaluated. Fifty-nine of 78 (75.64%) tumors showed nuclear staining for brachyury, and among them, 29 tumors (49.15%) showed 1+ (<30% positive cells) staining, 15 tumors (25.42%) had 2+ (31% to 60% positive cells) staining, and 15 tumors (25.42%) demonstrated 3+ (61% to 100% positive cells) staining. Brachyury nuclear staining was detected more frequently in sacral chordomas than in chordomas of the mobile spine. However, there was no significant relationship between brachyury expression and other clinical variables. By Kaplan-Meier analysis, brachyury expression failed to produce any significant relationship with the overall survival rate. In conclusion, brachyury expression is not a prognostic indicator in chordoma.

Comparison of gene expression responses to hypoxia in viviparous (Xiphophorus) and oviparous (Oryzias) fishes using a medaka microarray.

PubMed

Boswell, Mikki G; Wells, Melissa C; Kirk, Lyndsey M; Ju, Zhenlin; Zhang, Ziping; Booth, Rachell E; Walter, Ronald B

2009-03-01

Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka (Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species (Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant (p<0.05)+/-3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 x 10(-5)) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).

Identification of Novel Tissue-Specific Genes by Analysis of Microarray Databases: A Human and Mouse Model

PubMed Central

Suh, Yeunsu; Davis, Michael E.; Lee, Kichoon

2013-01-01

Understanding the tissue-specific pattern of gene expression is critical in elucidating the molecular mechanisms of tissue development, gene function, and transcriptional regulations of biological processes. Although tissue-specific gene expression information is available in several databases, follow-up strategies to integrate and use these data are limited. The objective of the current study was to identify and evaluate novel tissue-specific genes in human and mouse tissues by performing comparative microarray database analysis and semi-quantitative PCR analysis. We developed a powerful approach to predict tissue-specific genes by analyzing existing microarray data from the NCBI′s Gene Expression Omnibus (GEO) public repository. We investigated and confirmed tissue-specific gene expression in the human and mouse kidney, liver, lung, heart, muscle, and adipose tissue. Applying our novel comparative microarray approach, we confirmed 10 kidney, 11 liver, 11 lung, 11 heart, 8 muscle, and 8 adipose specific genes. The accuracy of this approach was further verified by employing semi-quantitative PCR reaction and by searching for gene function information in existing publications. Three novel tissue-specific genes were discovered by this approach including AMDHD1 (amidohydrolase domain containing 1) in the liver, PRUNE2 (prune homolog 2) in the heart, and ACVR1C (activin A receptor, type IC) in adipose tissue. We further confirmed the tissue-specific expression of these 3 novel genes by real-time PCR. Among them, ACVR1C is adipose tissue-specific and adipocyte-specific in adipose tissue, and can be used as an adipocyte developmental marker. From GEO profiles, we predicted the processes in which AMDHD1 and PRUNE2 may participate. Our approach provides a novel way to identify new sets of tissue-specific genes and to predict functions in which they may be involved. PMID:23741331

Genome-wide prediction and analysis of human tissue-selective genes using microarray expression data

PubMed Central

2013-01-01

Background Understanding how genes are expressed specifically in particular tissues is a fundamental question in developmental biology. Many tissue-specific genes are involved in the pathogenesis of complex human diseases. However, experimental identification of tissue-specific genes is time consuming and difficult. The accurate predictions of tissue-specific gene targets could provide useful information for biomarker development and drug target identification. Results In this study, we have developed a machine learning approach for predicting the human tissue-specific genes using microarray expression data. The lists of known tissue-specific genes for different tissues were collected from UniProt database, and the expression data retrieved from the previously compiled dataset according to the lists were used for input vector encoding. Random Forests (RFs) and Support Vector Machines (SVMs) were used to construct accurate classifiers. The RF classifiers were found to outperform SVM models for tissue-specific gene prediction. The results suggest that the candidate genes for brain or liver specific expression can provide valuable information for further experimental studies. Our approach was also applied for identifying tissue-selective gene targets for different types of tissues. Conclusions A machine learning approach has been developed for accurately identifying the candidate genes for tissue specific/selective expression. The approach provides an efficient way to select some interesting genes for developing new biomedical markers and improve our knowledge of tissue-specific expression. PMID:23369200

Cruella: developing a scalable tissue microarray data management system.

PubMed

Cowan, James D; Rimm, David L; Tuck, David P

2006-06-01

Compared with DNA microarray technology, relatively little information is available concerning the special requirements, design influences, and implementation strategies of data systems for tissue microarray technology. These issues include the requirement to accommodate new and different data elements for each new project as well as the need to interact with pre-existing models for clinical, biological, and specimen-related data. To design and implement a flexible, scalable tissue microarray data storage and management system that could accommodate information regarding different disease types and different clinical investigators, and different clinical investigation questions, all of which could potentially contribute unforeseen data types that require dynamic integration with existing data. The unpredictability of the data elements combined with the novelty of automated analysis algorithms and controlled vocabulary standards in this area require flexible designs and practical decisions. Our design includes a custom Java-based persistence layer to mediate and facilitate interaction with an object-relational database model and a novel database schema. User interaction is provided through a Java Servlet-based Web interface. Cruella has become an indispensable resource and is used by dozens of researchers every day. The system stores millions of experimental values covering more than 300 biological markers and more than 30 disease types. The experimental data are merged with clinical data that has been aggregated from multiple sources and is available to the researchers for management, analysis, and export. Cruella addresses many of the special considerations for managing tissue microarray experimental data and the associated clinical information. A metadata-driven approach provides a practical solution to many of the unique issues inherent in tissue microarray research, and allows relatively straightforward interoperability with and accommodation of new data models.

Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

PubMed Central

von Schalburg, Kristian R; Rise, Matthew L; Cooper, Glenn A; Brown, Gordon D; Gibbs, A Ross; Nelson, Colleen C; Davidson, William S; Koop, Ben F

2005-01-01

Background We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. Results We have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA. Conclusion We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content. PMID:16164747

Microarray expression profiling in adhesion and normal peritoneal tissues.

PubMed

Ambler, Dana R; Golden, Alicia M; Gell, Jennifer S; Saed, Ghassan M; Carey, David J; Diamond, Michael P

2012-05-01

To identify molecular markers associated with adhesion and normal peritoneal tissue using microarray expression profiling. Comparative study. University hospital. Five premenopausal women. Adhesion and normal peritoneal tissue samples were obtained from premenopausal women. Ribonucleic acid was extracted using standard protocols and processed for hybridization to Affymetrix Whole Transcript Human Gene Expression Chips. Microarray data were obtained from five different patients, each with adhesion tissue and normal peritoneal samples. Real-time polymerase chain reaction was performed for confirmation using standard protocols. Gene expression in postoperative adhesion and normal peritoneal tissues. A total of 1,263 genes were differentially expressed between adhesion and normal tissues. One hundred seventy-three genes were found to be up-regulated and 56 genes were down-regulated in the adhesion tissues compared with normal peritoneal tissues. The genes were sorted into functional categories according to Gene Ontology annotations. Twenty-six up-regulated genes and 11 down-regulated genes were identified with functions potentially relevant to the pathophysiology of postoperative adhesions. We evaluated and confirmed expression of 12 of these specific genes via polymerase chain reaction. The pathogenesis, natural history, and optimal treatment of postoperative adhesive disease remains unanswered. Microarray analysis of adhesions identified specific genes with increased and decreased expression when compared with normal peritoneum. Knowledge of these genes and ontologic pathways with altered expression provide targets for new therapies to treat patients who have or are at risk for postoperative adhesions. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

PubMed Central

2013-01-01

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms. PMID:24160452

Finding Patterns of Emergence in Science and Technology

DTIC Science & Technology

2012-09-24

formal evaluation scheduled – Case Studies, Eight Examples: Tissue Engineering, Cold Fusion, RF Metamaterials, DNA Microarrays, Genetic Algorithms, RNAi...emerging capabilities Case Studies, Eight Examples: • Tissue Engineering, Cold Fusion, RF Metamaterials, DNA Microarrays, Genetic Algorithms...Evidence Quality (i.e., the rubric ) and deliver comprehensible evidential support for nomination • Demonstrate proof-of-concept nomination for Chinese

Decentralized Data Sharing of Tissue Microarrays for Investigative Research in Oncology

PubMed Central

Chen, Wenjin; Schmidt, Cristina; Parashar, Manish; Reiss, Michael; Foran, David J.

2007-01-01

Tissue microarray technology (TMA) is a relatively new approach for efficiently and economically assessing protein and gene expression across large ensembles of tissue specimens. Tissue microarray technology holds great potential for reducing the time and cost associated with conducting research in tissue banking, proteomics, and outcome studies. However, the sheer volume of images and other data generated from even limited studies involving tissue microarrays quickly approach the processing capacity and resources of a division or department. This challenge is compounded by the fact that large-scale projects in several areas of modern research rely upon multi-institutional efforts in which investigators and resources are spread out over multiple campuses, cities, and states. To address some of the data management issues several leading institutions have begun to develop their own “in-house” systems, independently, but such data will be only minimally useful if it isn’t accessible to others in the scientific community. Investigators at different institutions studying the same or related disorders might benefit from the synergy of sharing results. To facilitate sharing of TMA data across different database implementations, the Technical Standards Committee of the Association for Pathology Informatics organized workshops in efforts to establish a standardized TMA data exchange specification. The focus of our research does not relate to the establishment of standards for exchange, but rather builds on these efforts and concentrates on the design, development and deployment of a decentralized collaboratory for the unsupervised characterization, and seamless and secure discovery and sharing of TMA data. Specifically, we present a self-organizing, peer-to-peer indexing and discovery infrastructure for quantitatively assessing digitized TMA’s. The system utilizes a novel, optimized decentralized search engine that supports flexible querying, while guaranteeing that once information has been stored in the system, it will be found with bounded costs. PMID:19081778

MGDB: crossing the marker genes of a user microarray with a database of public-microarrays marker genes.

PubMed

Huerta, Mario; Munyi, Marc; Expósito, David; Querol, Enric; Cedano, Juan

2014-06-15

The microarrays performed by scientific teams grow exponentially. These microarray data could be useful for researchers around the world, but unfortunately they are underused. To fully exploit these data, it is necessary (i) to extract these data from a repository of the high-throughput gene expression data like Gene Expression Omnibus (GEO) and (ii) to make the data from different microarrays comparable with tools easy to use for scientists. We have developed these two solutions in our server, implementing a database of microarray marker genes (Marker Genes Data Base). This database contains the marker genes of all GEO microarray datasets and it is updated monthly with the new microarrays from GEO. Thus, researchers can see whether the marker genes of their microarray are marker genes in other microarrays in the database, expanding the analysis of their microarray to the rest of the public microarrays. This solution helps not only to corroborate the conclusions regarding a researcher's microarray but also to identify the phenotype of different subsets of individuals under investigation, to frame the results with microarray experiments from other species, pathologies or tissues, to search for drugs that promote the transition between the studied phenotypes, to detect undesirable side effects of the treatment applied, etc. Thus, the researcher can quickly add relevant information to his/her studies from all of the previous analyses performed in other studies as long as they have been deposited in public repositories. Marker-gene database tool: http://ibb.uab.es/mgdb © The Author 2014. Published by Oxford University Press.

Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples.

PubMed

Pine, P S; Boedigheimer, M; Rosenzweig, B A; Turpaz, Y; He, Y D; Delenstarr, G; Ganter, B; Jarnagin, K; Jones, W D; Reid, L H; Thompson, K L

2008-11-01

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

Soy consumption and histopathologic markers in breast tissue using tissue microarrays.

PubMed

Maskarinec, Gertraud; Erber, Eva; Verheus, Martijn; Hernandez, Brenda Y; Killeen, Jeffrey; Cashin, Suzanne; Cline, J Mark

2009-01-01

This study examined the relation of soy intake with hormonal and proliferation markers in benign and malignant breast tissue using tissue microarrays (TMAs). TMAs with up to 4 malignant and 4 benign tissue samples for 268 breast cancer cases were constructed. Soy intake in early life and in adulthood was assessed by questionnaire. The TMAs were stained for estrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), proliferating cell nuclear antigen (PCNA), and Ki-67 using standard immunohistochemical methods. Logistic regression was applied for statistical analysis. A higher percentage of women showed positive marker expression in malignant than in benign tissue. With one exception, HER2/neu, no significant associations between soy intake and pathologic markers were observed. Early life soy intake was associated with lower HER2/neu and PCNA staining of malignant tissue. In benign tissue, early life soy intake showed higher ER and PR expression, but no difference in proliferation markers. The results of this investigation provide some assurance that soy intake does not adversely affect markers of proliferation. TMAs were shown to be a useful tool for epidemiologic research.

The tissue microarray data exchange specification: Extending TMA DES to provide flexible scoring and incorporate virtual slides

PubMed Central

Wright, Alexander; Lyttleton, Oliver; Lewis, Paul; Quirke, Philip; Treanor, Darren

2011-01-01

Background: Tissue MicroArrays (TMAs) are a high throughput technology for rapid analysis of protein expression across hundreds of patient samples. Often, data relating to TMAs is specific to the clinical trial or experiment it is being used for, and not interoperable. The Tissue Microarray Data Exchange Specification (TMA DES) is a set of eXtensible Markup Language (XML)-based protocols for storing and sharing digitized Tissue Microarray data. XML data are enclosed by named tags which serve as identifiers. These tag names can be Common Data Elements (CDEs), which have a predefined meaning or semantics. By using this specification in a laboratory setting with increasing demands for digital pathology integration, we found that the data structure lacked the ability to cope with digital slide imaging in respect to web-enabled digital pathology systems and advanced scoring techniques. Materials and Methods: By employing user centric design, and observing behavior in relation to TMA scoring and associated data, the TMA DES format was extended to accommodate the current limitations. This was done with specific focus on developing a generic tool for handling any given scoring system, and utilizing data for multiple observations and observers. Results: DTDs were created to validate the extensions of the TMA DES protocol, and a test set of data containing scores for 6,708 TMA core images was generated. The XML was then read into an image processing algorithm to utilize the digital pathology data extensions, and scoring results were easily stored alongside the existing multiple pathologist scores. Conclusions: By extending the TMA DES format to include digital pathology data and customizable scoring systems for TMAs, the new system facilitates the collaboration between pathologists and organizations, and can be used in automatic or manual data analysis. This allows complying systems to effectively communicate complex and varied scoring data. PMID:21572508

Unique Chemokine Profiles of Lung Tissues Distinguish Post-chemotherapeutic Persistent and Chronic Tuberculosis in a Mouse Model.

PubMed

Park, Soomin; Baek, Seung-Hun; Cho, Sang-Nae; Jang, Young-Saeng; Kim, Ahreum; Choi, In-Hong

2017-01-01

There is a substantial need for biomarkers to distinguish latent stage from active Mycobacterium tuberculosis infections, for predicting disease progression. To induce the reactivation of tuberculosis, we present a new experimental animal model modified based on the previous model established by our group. In the new model, the reactivation of tuberculosis is induced without administration of immunosuppressive agents, which might disturb immune responses. To identify the immunological status of the persistent and chronic stages, we analyzed immunological genes in lung tissues from mice infected with M. tuberculosis . Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR. Based on the cDNA microarray results, 11 candidate cytokines genes, which were obviously up-regulated during the chronic stage compared with those during the persistent stage, were selected and clustered into three groups: (1) chemokine genes, except those of monocyte chemoattractant proteins (MCPs; CXCL9, CXCL10, CXCL11, CCL5, CCL19); (2) MCP genes (CCL2, CCL7, CCL8, CCL12); and (3) TNF and IFN-γ genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that the mRNA expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the persistent stage. Three chemokines (CCL5, CCL19, and CXCL9) and three MCPs (CCL7, CCL2, and CCL12) were noticeably increased in the chronic stage compared with the persistent stage by cDNA microarray ( p < 0.01, except CCL12) or RT-PCR ( p < 0.01). Therefore, these six significantly increased cytokines in lung tissue from the mouse tuberculosis model might be candidates for biomarkers to distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient tuberculosis markers.

Molecular profiles of pre- and postoperative breast cancer tumours reveal differentially expressed genes.

PubMed

Riis, Margit L H; Lüders, Torben; Markert, Elke K; Haakensen, Vilde D; Nesbakken, Anne-Jorun; Kristensen, Vessela N; Bukholm, Ida R K

2012-01-01

Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun families as well as genes of which the expression has previously been associated with cancer. The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology.

Molecular Profiles of Pre- and Postoperative Breast Cancer Tumours Reveal Differentially Expressed Genes

PubMed Central

Riis, Margit L. H.; Lüders, Torben; Markert, Elke K.; Haakensen, Vilde D.; Nesbakken, Anne-Jorun; Kristensen, Vessela N.; Bukholm, Ida R. K.

2012-01-01

Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun families as well as genes of which the expression has previously been associated with cancer. The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology. PMID:23227362

TAMEE: data management and analysis for tissue microarrays.

PubMed

Thallinger, Gerhard G; Baumgartner, Kerstin; Pirklbauer, Martin; Uray, Martina; Pauritsch, Elke; Mehes, Gabor; Buck, Charles R; Zatloukal, Kurt; Trajanoski, Zlatko

2007-03-07

With the introduction of tissue microarrays (TMAs) researchers can investigate gene and protein expression in tissues on a high-throughput scale. TMAs generate a wealth of data calling for extended, high level data management. Enhanced data analysis and systematic data management are required for traceability and reproducibility of experiments and provision of results in a timely and reliable fashion. Robust and scalable applications have to be utilized, which allow secure data access, manipulation and evaluation for researchers from different laboratories. TAMEE (Tissue Array Management and Evaluation Environment) is a web-based database application for the management and analysis of data resulting from the production and application of TMAs. It facilitates storage of production and experimental parameters, of images generated throughout the TMA workflow, and of results from core evaluation. Database content consistency is achieved using structured classifications of parameters. This allows the extraction of high quality results for subsequent biologically-relevant data analyses. Tissue cores in the images of stained tissue sections are automatically located and extracted and can be evaluated using a set of predefined analysis algorithms. Additional evaluation algorithms can be easily integrated into the application via a plug-in interface. Downstream analysis of results is facilitated via a flexible query generator. We have developed an integrated system tailored to the specific needs of research projects using high density TMAs. It covers the complete workflow of TMA production, experimental use and subsequent analysis. The system is freely available for academic and non-profit institutions from http://genome.tugraz.at/Software/TAMEE.

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

PubMed

Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

2017-01-12

Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

Data-adaptive test statistics for microarray data.

PubMed

Mukherjee, Sach; Roberts, Stephen J; van der Laan, Mark J

2005-09-01

An important task in microarray data analysis is the selection of genes that are differentially expressed between different tissue samples, such as healthy and diseased. However, microarray data contain an enormous number of dimensions (genes) and very few samples (arrays), a mismatch which poses fundamental statistical problems for the selection process that have defied easy resolution. In this paper, we present a novel approach to the selection of differentially expressed genes in which test statistics are learned from data using a simple notion of reproducibility in selection results as the learning criterion. Reproducibility, as we define it, can be computed without any knowledge of the 'ground-truth', but takes advantage of certain properties of microarray data to provide an asymptotically valid guide to expected loss under the true data-generating distribution. We are therefore able to indirectly minimize expected loss, and obtain results substantially more robust than conventional methods. We apply our method to simulated and oligonucleotide array data. By request to the corresponding author.

Expression profile of circular RNAs in human gastric cancer tissues

PubMed Central

Huang, You-Sheng; Jie, Na; Zou, Ke-Jian; Weng, Yang

2017-01-01

Circular RNAs (circRNAs) represent a newly identified class of non-coding RNA molecules, which interfere with gene transcription by adsorbing microRNAs (miRNAs). CircRNAs serve important roles in disease development and have the potential to serve as a novel class of biomarkers for clinical diagnosis. However, the role of circRNAs in the occurrence and development of gastric cancer (GC) remains unclear. In the present study, the expression profiles of circRNAs were compared between GC and adjacent normal tissues using a circRNA microarray, following which quantitative polymerase chain reaction (qPCR) was used to confirm the results of the circRNA microarray. Compared with the adjacent, normal mucosal tissues, 16 circRNAs were upregulated and 84 circRNAs were downregulated in GC. A total of 10 circRNAs were selected for validation in three pairs of GC and adjacent noncancerous tissues. The qPCR results were consistent with the findings of the microarray-based expression analysis. Of the circRNAs studied, only circRNA-0026 (hsa_circ_0000026) exhibited significantly different expression in GC (2.8-fold, P=0.001). Furthermore, online Database for Annotation, Visualization and Integrated Discovery annotation was used to predict circRNA-targeted miRNA-gene interactions. The analysis revealed that circRNA-0026 may regulate RNA transcription, RNA metabolism, gene expression, gene silencing and other biological functions in GC. In conclusion, differential expression of circRNAs may be associated with GC tumorigenesis, and circRNA-0026 is a promising biomarker for GC diagnosis and targeted therapy. PMID:28737829

Microarray Meta-Analysis of RNA-Binding Protein Functions in Alternative Polyadenylation

PubMed Central

Hu, Wenchao; Liu, Yuting; Yan, Jun

2014-01-01

Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community. PMID:24622240

Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array

PubMed Central

Wong, Yick Ching; Kwong, Qi Bin; Lee, Heng Leng; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R.; Kulaveerasingam, Harikrishna

2014-01-01

Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA) and triacylglycerol (TAG) assembly, along with the tricarboxylic acid cycle (TCA) and glycolysis pathway at 16 Weeks After Anthesis (WAA) exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01), and rice (p-value < 0.01) arrays. The oil palm microarray data also showed comparable correlation of expression (r2 = 0.569, p < 0.01) throughout mesocarp development to transcriptome (RNA sequencing) data, and improved correlation over quantitative real-time PCR (qPCR) (r2 = 0.721, p < 0.01) of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield. PMID:27600348

Expression Comparison of Oil Biosynthesis Genes in Oil Palm Mesocarp Tissue Using Custom Array.

PubMed

Wong, Yick Ching; Kwong, Qi Bin; Lee, Heng Leng; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R; Kulaveerasingam, Harikrishna

2014-11-13

Gene expression changes that occur during mesocarp development are a major research focus in oil palm research due to the economic importance of this tissue and the relatively rapid increase in lipid content to very high levels at fruit ripeness. Here, we report the development of a transcriptome-based 105,000-probe oil palm mesocarp microarray. The expression of genes involved in fatty acid (FA) and triacylglycerol (TAG) assembly, along with the tricarboxylic acid cycle (TCA) and glycolysis pathway at 16 Weeks After Anthesis (WAA) exhibited significantly higher signals compared to those obtained from a cross-species hybridization to the Arabidopsis (p-value < 0.01), and rice (p-value < 0.01) arrays. The oil palm microarray data also showed comparable correlation of expression (r² = 0.569, p < 0.01) throughout mesocarp development to transcriptome (RNA sequencing) data, and improved correlation over quantitative real-time PCR (qPCR) (r² = 0.721, p < 0.01) of the same RNA samples. The results confirm the advantage of the custom microarray over commercially available arrays derived from model species. We demonstrate the utility of this custom microarray to gain a better understanding of gene expression patterns in the oil palm mesocarp that may lead to increasing future oil yield.

Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis

PubMed Central

Wapner, Ronald J.; Martin, Christa Lese; Levy, Brynn; Ballif, Blake C.; Eng, Christine M.; Zachary, Julia M.; Savage, Melissa; Platt, Lawrence D.; Saltzman, Daniel; Grobman, William A.; Klugman, Susan; Scholl, Thomas; Simpson, Joe Leigh; McCall, Kimberly; Aggarwal, Vimla S.; Bunke, Brian; Nahum, Odelia; Patel, Ankita; Lamb, Allen N.; Thom, Elizabeth A.; Beaudet, Arthur L.; Ledbetter, David H.; Shaffer, Lisa G.; Jackson, Laird

2013-01-01

Background Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Methods Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. Results We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down’s syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. Conclusions In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.) PMID:23215555

Testing an aflatoxin B1 gene signature in rat archival tissues.

PubMed

Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

2012-05-21

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.

DNA microarrays: a powerful genomic tool for biomedical and clinical research

PubMed Central

Trevino, Victor; Falciani, Francesco; Barrera-Saldaña, Hugo A.

2007-01-01

Among the many benefits of the Human Genome Project are new and powerful tools such as the genome-wide hybridization devices referred as microarrays. Initially designed to measure gene transcriptional levels, microarray technologies are now used for comparing other genome features among individuals and their tissues and cells. Results provide valuable information on disease subcategories, disease prognosis, and treatment outcome. Likewise, reveal differences in genetic makeup, regulatory mechanisms and subtle variations are approaching the era of personalized medicine. To understand this powerful tool, its versatility and how it is dramatically changing the molecular approach to biomedical and clinical research, this review describes the technology, its applications, a didactic step-by-step review of a typical microarray protocol, and a real experiment. Finally, it calls the attention of the medical community to integrate multidisciplinary teams, to take advantage of this technology and its expanding applications that in a slide reveals our genetic inheritance and destiny. PMID:17660860

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways. PMID:16608515

Rapid Spoligotyping of Mycobacterium tuberculosis Complex Bacteria by Use of a Microarray System with Automatic Data Processing and Assignment

PubMed Central

Ruettger, Anke; Nieter, Johanna; Skrypnyk, Artem; Engelmann, Ines; Ziegler, Albrecht; Moser, Irmgard; Monecke, Stefan; Ehricht, Ralf

2012-01-01

Membrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assay's validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability. PMID:22553239

Rapid spoligotyping of Mycobacterium tuberculosis complex bacteria by use of a microarray system with automatic data processing and assignment.

PubMed

Ruettger, Anke; Nieter, Johanna; Skrypnyk, Artem; Engelmann, Ines; Ziegler, Albrecht; Moser, Irmgard; Monecke, Stefan; Ehricht, Ralf; Sachse, Konrad

2012-07-01

Membrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assay's validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability.

TIPMaP: a web server to establish transcript isoform profiles from reliable microarray probes.

PubMed

Chitturi, Neelima; Balagannavar, Govindkumar; Chandrashekar, Darshan S; Abinaya, Sadashivam; Srini, Vasan S; Acharya, Kshitish K

2013-12-27

Standard 3' Affymetrix gene expression arrays have contributed a significantly higher volume of existing gene expression data than other microarray platforms. These arrays were designed to identify differentially expressed genes, but not their alternatively spliced transcript forms. No resource can currently identify expression pattern of specific mRNA forms using these microarray data, even though it is possible to do this. We report a web server for expression profiling of alternatively spliced transcripts using microarray data sets from 31 standard 3' Affymetrix arrays for human, mouse and rat species. The tool has been experimentally validated for mRNAs transcribed or not-detected in a human disease condition (non-obstructive azoospermia, a male infertility condition). About 4000 gene expression datasets were downloaded from a public repository. 'Good probes' with complete coverage and identity to latest reference transcript sequences were first identified. Using them, 'Transcript specific probe-clusters' were derived for each platform and used to identify expression status of possible transcripts. The web server can lead the user to datasets corresponding to specific tissues, conditions via identifiers of the microarray studies or hybridizations, keywords, official gene symbols or reference transcript identifiers. It can identify, in the tissues and conditions of interest, about 40% of known transcripts as 'transcribed', 'not-detected' or 'differentially regulated'. Corresponding additional information for probes, genes, transcripts and proteins can be viewed too. We identified the expression of transcripts in a specific clinical condition and validated a few of these transcripts by experiments (using reverse transcription followed by polymerase chain reaction). The experimental observations indicated higher agreements with the web server results, than contradictions. The tool is accessible at http://resource.ibab.ac.in/TIPMaP. The newly developed online tool forms a reliable means for identification of alternatively spliced transcript-isoforms that may be differentially expressed in various tissues, cell types or physiological conditions. Thus, by making better use of existing data, TIPMaP avoids the dependence on precious tissue-samples, in experiments with a goal to establish expression profiles of alternative splice forms--at least in some cases.

Expression analysis of URI/RMP gene in endometrioid adenocarcinoma by tissue microarray immunohistochemistry.

PubMed

Gu, Junxia; Liang, Yuting; Qiao, Longwei; Li, Xiaoyun; Li, Xingang; Lu, Yaojuan; Zheng, Qiping

2013-01-01

Multiple studies have recently demonstrated the oncogenic property of URI (or RMP, a member of the prefoldin family of molecular chaperones) during progression of hepatocellular carcinoma, ovarian cancer, and possibly prostate cancer. Most recently, we have shown that URI/RMP is up-regulated in cervical cancer, another reproductive system tumor beside ovarian and prostate cancers. To investigate if URI/RMP also plays a role in other reproductive system tumors, especially in endometrioid adenocarcinoma, we analyzed URI/RMP expression in a TMA (tissue microarray) containing tissues from 30 cases of endometrioid adenocarcinoma (which covers tumor tissues from Grade I through Grade III) and adjacent endometrium by immunohistochemistry (IHC) and densitometry analysis using image-pro plus 6.0 software. Our results showed that the mean density of URI/RMP expression in cancerous tissue is slightly higher than that of the adjacent endometrial tissue, though not statistically significant (p>0.05). There is no significant difference either between the mean density of Grade III cancerous tissue and that of Grade I and II cancers. Notably, we detected significantly higher signal intensity in cancerous tissue of all 7 Grade III cases than that of their adjacent endometrial tissue (p<0.05), suggesting a correlation of URI/RMP expression with the differentiation and pathological classification of endometrioid adenocarcinoma. Together, our results demonstrate the heterogeneous expression of URI/RMP in endometrioid adenocarcinoma. The higher level of URI/RMP expression in high-grade endometrioid adenocarcinomas compared to tissues of adjacent endometrium or gland suggests a diagnostic and possibly, a prognostic value of URI/RMP in endometrioid adenocarcinoma.

Expression analysis of URI/RMP gene in endometrioid adenocarcinoma by tissue microarray immunohistochemistry

PubMed Central

Gu, Junxia; Liang, Yuting; Qiao, Longwei; Li, Xiaoyun; Li, Xingang; Lu, Yaojuan; Zheng, Qiping

2013-01-01

Multiple studies have recently demonstrated the oncogenic property of URI (or RMP, a member of the prefoldin family of molecular chaperones) during progression of hepatocellular carcinoma, ovarian cancer, and possibly prostate cancer. Most recently, we have shown that URI/RMP is up-regulated in cervical cancer, another reproductive system tumor beside ovarian and prostate cancers. To investigate if URI/RMP also plays a role in other reproductive system tumors, especially in endometrioid adenocarcinoma, we analyzed URI/RMP expression in a TMA (tissue microarray) containing tissues from 30 cases of endometrioid adenocarcinoma (which covers tumor tissues from Grade I through Grade III) and adjacent endometrium by immunohistochemistry (IHC) and densitometry analysis using image-pro plus 6.0 software. Our results showed that the mean density of URI/RMP expression in cancerous tissue is slightly higher than that of the adjacent endometrial tissue, though not statistically significant (p>0.05). There is no significant difference either between the mean density of Grade III cancerous tissue and that of Grade I and II cancers. Notably, we detected significantly higher signal intensity in cancerous tissue of all 7 Grade III cases than that of their adjacent endometrial tissue (p<0.05), suggesting a correlation of URI/RMP expression with the differentiation and pathological classification of endometrioid adenocarcinoma. Together, our results demonstrate the heterogeneous expression of URI/RMP in endometrioid adenocarcinoma. The higher level of URI/RMP expression in high-grade endometrioid adenocarcinomas compared to tissues of adjacent endometrium or gland suggests a diagnostic and possibly, a prognostic value of URI/RMP in endometrioid adenocarcinoma. PMID:24228101

A metadata-aware application for remote scoring and exchange of tissue microarray images

PubMed Central

2013-01-01

Background The use of tissue microarrays (TMA) and advances in digital scanning microscopy has enabled the collection of thousands of tissue images. There is a need for software tools to annotate, query and share this data amongst researchers in different physical locations. Results We have developed an open source web-based application for remote scoring of TMA images, which exploits the value of Microsoft Silverlight Deep Zoom to provide a intuitive interface for zooming and panning around digital images. We use and extend existing XML-based standards to ensure that the data collected can be archived and that our system is interoperable with other standards-compliant systems. Conclusion The application has been used for multi-centre scoring of TMA slides composed of tissues from several Phase III breast cancer trials and ten different studies participating in the International Breast Cancer Association Consortium (BCAC). The system has enabled researchers to simultaneously score large collections of TMA and export the standardised data to integrate with pathological and clinical outcome data, thereby facilitating biomarker discovery. PMID:23635078

Validation of the Swine Protein-Annotated Oligonucleotide Microarray

USDA-ARS?s Scientific Manuscript database

The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

Cardiac mesenchymal progenitors from postmortem cardiac tissues retained cellular characterization.

PubMed

Kami, D; Kitani, T; Nakata, M; Gojo, S

2014-05-01

Currently, cells for transplantation in regenerative medicine are derived from either autologous or allogeneic tissue. The former has the drawbacks that the quality of donor cells may depend on the condition of the patient, while the quantity of the cells may also be limited. To solve these problems, we investigated the potential of allogeneic cardiac mesenchymal progenitors (CMPs) derived from postmortem hearts, which may be immunologically privileged similar to bone marrow-derived mesenchymal progenitors. We examined whether viable CMPs could be isolated from C57/B6 murine cardiac tissues harvested at 24 hours postmortem. After 2- to 3-week propagation with a high dose of basic fibroblast growth factor, we performed cellular characteristics analyses, which included proliferation and differentiation property flow cytometry and microarray analyses. Postmortem CMPs had a longer lag phase after seeding than CMPs obtained from living tissues, but otherwise had similar characteristics in all the analyses. In addition, global gene expression analysis by microarray showed that cells derived from postmortem and living tissues had similar characteristics. These results indicate that allogeneic postmortem CMPs have potential for cell transplantation because they circumvent the issue of both the quality and quantity of donor cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Novel statistical framework to identify differentially expressed genes allowing transcriptomic background differences.

PubMed

Ling, Zhi-Qiang; Wang, Yi; Mukaisho, Kenichi; Hattori, Takanori; Tatsuta, Takeshi; Ge, Ming-Hua; Jin, Li; Mao, Wei-Min; Sugihara, Hiroyuki

2010-06-01

Tests of differentially expressed genes (DEGs) from microarray experiments are based on the null hypothesis that genes that are irrelevant to the phenotype/stimulus are expressed equally in the target and control samples. However, this strict hypothesis is not always true, as there can be several transcriptomic background differences between target and control samples, including different cell/tissue types, different cell cycle stages and different biological donors. These differences lead to increased false positives, which have little biological/medical significance. In this article, we propose a statistical framework to identify DEGs between target and control samples from expression microarray data allowing transcriptomic background differences between these samples by introducing a modified null hypothesis that the gene expression background difference is normally distributed. We use an iterative procedure to perform robust estimation of the null hypothesis and identify DEGs as outliers. We evaluated our method using our own triplicate microarray experiment, followed by validations with reverse transcription-polymerase chain reaction (RT-PCR) and on the MicroArray Quality Control dataset. The evaluations suggest that our technique (i) results in less false positive and false negative results, as measured by the degree of agreement with RT-PCR of the same samples, (ii) can be applied to different microarray platforms and results in better reproducibility as measured by the degree of DEG identification concordance both intra- and inter-platforms and (iii) can be applied efficiently with only a few microarray replicates. Based on these evaluations, we propose that this method not only identifies more reliable and biologically/medically significant DEG, but also reduces the power-cost tradeoff problem in the microarray field. Source code and binaries freely available for download at http://comonca.org.cn/fdca/resources/softwares/deg.zip.

Differential Adipose Tissue Gene Expression Profiles in Abacavir Treated Patients That May Contribute to the Understanding of Cardiovascular Risk: A Microarray Study

PubMed Central

Shahmanesh, Mohsen; Phillips, Kenneth; Boothby, Meg; Tomlinson, Jeremy W.

2015-01-01

Objective To compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofovir (TDF) or zidovidine (AZT). Design Subcutaneous fat biopsies were obtained before, at 6- and 18–24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis. Results There were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18–24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18–24 months (adjusted p<0.05) and focal adhesions and tight junction at 6 months (p<0.5). Genes controlling leukocyte transendothelial migration (p<0.05) and ECM-receptor interactions (p = 0.04) were over-expressed in ABC compared to TDF and AZT at 6 months but not at 18–24 months. Enrichment of pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group in comparison with other treatments. There was little difference between AZT and TDF. Conclusion After initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens. PMID:25617630

Semantically enabled and statistically supported biological hypothesis testing with tissue microarray databases

PubMed Central

2011-01-01

Background Although many biological databases are applying semantic web technologies, meaningful biological hypothesis testing cannot be easily achieved. Database-driven high throughput genomic hypothesis testing requires both of the capabilities of obtaining semantically relevant experimental data and of performing relevant statistical testing for the retrieved data. Tissue Microarray (TMA) data are semantically rich and contains many biologically important hypotheses waiting for high throughput conclusions. Methods An application-specific ontology was developed for managing TMA and DNA microarray databases by semantic web technologies. Data were represented as Resource Description Framework (RDF) according to the framework of the ontology. Applications for hypothesis testing (Xperanto-RDF) for TMA data were designed and implemented by (1) formulating the syntactic and semantic structures of the hypotheses derived from TMA experiments, (2) formulating SPARQLs to reflect the semantic structures of the hypotheses, and (3) performing statistical test with the result sets returned by the SPARQLs. Results When a user designs a hypothesis in Xperanto-RDF and submits it, the hypothesis can be tested against TMA experimental data stored in Xperanto-RDF. When we evaluated four previously validated hypotheses as an illustration, all the hypotheses were supported by Xperanto-RDF. Conclusions We demonstrated the utility of high throughput biological hypothesis testing. We believe that preliminary investigation before performing highly controlled experiment can be benefited. PMID:21342584

Micatu Tissue Arrayer | NCI Technology Transfer Center | TTC

Cancer.gov

An NCI researcher recognized a critical need to create a low-cost, easy-to-use tissue microarrayer (TMA), an instrument used by researchers and pathologists to accurately examine tissue samples from patients.

Upregulation of long non-coding RNA M26317 correlates with tumor progression and poor prognosis in gastric cancer.

PubMed

Li, Li; Wang, Yuan-Yu; Mou, Xiao Zhou; Ye, Zai-Yuan; Zhao, Zhong-Sheng

2018-04-23

To investigate the expression and clinical significance of long non-coding RNA (lnc RNA) in gastric cancer, we applied microarray analysis to obtain expression profiles of protein coding genes and lncRNAs in tumor and paired adjacent non-tumor tissues. We found that 41 lncRNAs were upregulated and 31 lncRNAs were downregulated more than 2-fold in gastric cancer versus noncancerous tissues (ratio>2.0, P<.01). We established a co-expression network of the differentially expressed lncRNAs and targeted coding genes that included 17 lncRNAs and 16 coding genes. As the results of microarray analysis showed that lncRNA M26317 was upregulated in gastric cancer tissues we examined the expression level of M26317 in 103 gastric cancer tissues by RT-PCR and 436 gastric cancer tissues by in situ hybridization. Our data confirmed that M26317 was upregulated in gastric cancer tissues. Moreover, expression of M26317 correlated with patient age, size of tumor, Lauren's classification, depth of invasion, lymph node and distant metastasis, TNM stage and poor prognosis (P<.05), but was not associated with gender, location of tumor, and differentiation (P>.05). M26317 may have an important role in malignant transformation and metastasis of gastric cancer. Copyright © 2018. Published by Elsevier Inc.

Transcriptome-Wide Mega-Analyses Reveal Joint Dysregulation of Immunologic Genes and Transcription Regulators in Brain and Blood in Schizophrenia

PubMed Central

Hess, Jonathan L.; Tylee, Daniel S.; Barve, Rahul; de Jong, Simone; Ophoff, Roel A.; Kumarasinghe, Nishantha; Tooney, Paul; Schall, Ulrich; Gardiner, Erin; Beveridge, Natalie Jane; Scott, Rodney J.; Yasawardene, Surangi; Perera, Antionette; Mendis, Jayan; Carr, Vaughan; Kelly, Brian; Cairns, Murray; Tsuang, Ming T.; Glatt, Stephen J.

2016-01-01

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n = 315) and from ex-vivo blood tissues (n = 578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia. PMID:27450777

Transcriptome-wide mega-analyses reveal joint dysregulation of immunologic genes and transcription regulators in brain and blood in schizophrenia.

PubMed

Hess, Jonathan L; Tylee, Daniel S; Barve, Rahul; de Jong, Simone; Ophoff, Roel A; Kumarasinghe, Nishantha; Tooney, Paul; Schall, Ulrich; Gardiner, Erin; Beveridge, Natalie Jane; Scott, Rodney J; Yasawardene, Surangi; Perera, Antionette; Mendis, Jayan; Carr, Vaughan; Kelly, Brian; Cairns, Murray; Tsuang, Ming T; Glatt, Stephen J

2016-10-01

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n=315) and from ex-vivo blood tissues (n=578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia. Published by Elsevier B.V.

Multi-Tissue Computational Modeling Analyzes Pathophysiology of Type 2 Diabetes in MKR Mice

PubMed Central

Kumar, Amit; Harrelson, Thomas; Lewis, Nathan E.; Gallagher, Emily J.; LeRoith, Derek; Shiloach, Joseph; Betenbaugh, Michael J.

2014-01-01

Computational models using metabolic reconstructions for in silico simulation of metabolic disorders such as type 2 diabetes mellitus (T2DM) can provide a better understanding of disease pathophysiology and avoid high experimentation costs. There is a limited amount of computational work, using metabolic reconstructions, performed in this field for the better understanding of T2DM. In this study, a new algorithm for generating tissue-specific metabolic models is presented, along with the resulting multi-confidence level (MCL) multi-tissue model. The effect of T2DM on liver, muscle, and fat in MKR mice was first studied by microarray analysis and subsequently the changes in gene expression of frank T2DM MKR mice versus healthy mice were applied to the multi-tissue model to test the effect. Using the first multi-tissue genome-scale model of all metabolic pathways in T2DM, we found out that branched-chain amino acids' degradation and fatty acids oxidation pathway is downregulated in T2DM MKR mice. Microarray data showed low expression of genes in MKR mice versus healthy mice in the degradation of branched-chain amino acids and fatty-acid oxidation pathways. In addition, the flux balance analysis using the MCL multi-tissue model showed that the degradation pathways of branched-chain amino acid and fatty acid oxidation were significantly downregulated in MKR mice versus healthy mice. Validation of the model was performed using data derived from the literature regarding T2DM. Microarray data was used in conjunction with the model to predict fluxes of various other metabolic pathways in the T2DM mouse model and alterations in a number of pathways were detected. The Type 2 Diabetes MCL multi-tissue model may explain the high level of branched-chain amino acids and free fatty acids in plasma of Type 2 Diabetic subjects from a metabolic fluxes perspective. PMID:25029527

A microarray analysis of sexual dimorphism of adipose tissues in high-fat-diet-induced obese mice

PubMed Central

Grove, KL; Fried, SK; Greenberg, AS; Xiao, XQ; Clegg, DJ

2013-01-01

Objective A sexual dimorphism exists in body fat distribution; females deposit relatively more fat in subcutaneous/inguinal depots whereas males deposit more fat in the intra-abdominal/gonadal depot. Our objective was to systematically document depot- and sex-related differences in the accumulation of adipose tissue and gene expression, comparing differentially expressed genes in diet-induced obese mice with mice maintained on a chow diet. Research Design and Methods We used a microarray approach to determine whether there are sexual dimorphisms in gene expression in age-matched male, female or ovariectomized female (OVX) C57/BL6 mice maintained on a high-fat (HF) diet. We then compared expression of validated genes between the sexes on a chow diet. Results After exposure to a high fat diet for 12 weeks, females gained less weight than males. The microarray analyses indicate in intra-abdominal/gonadal adipose tissue in females 1642 genes differ by at least twofold between the depots, whereas 706 genes differ in subcutaneous/inguinal adipose tissue when compared with males. Only 138 genes are commonly regulated in both sexes and adipose tissue depots. Inflammatory genes (cytokine–cytokine receptor interactions and acute-phase protein synthesis) are upregulated in males when compared with females, and there is a partial reversal after OVX, where OVX adipose tissue gene expression is more ′male-like′. This pattern is not observed in mice maintained on chow. Histology of male gonadal white adipose tissue (GWAT) shows more crown-like structures than females, indicative of inflammation and adipose tissue remodeling. In addition, genes related to insulin signaling and lipid synthesis are higher in females than males, regardless of dietary exposure. Conclusions These data suggest that male and female adipose tissue differ between the sexes regardless of diet. Moreover, HF diet exposure elicits a much greater inflammatory response in males when compared with females. This data set underscores the importance of analyzing depot-, sex- and steroid-dependent regulation of adipose tissue distribution and function. PMID:20157318

Immunohistochemical evidence of ubiquitous distribution of the metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines.

PubMed

Weirich, Gregor; Mengele, Karin; Yfanti, Christina; Gkazepis, Apostolos; Hellmann, Daniela; Welk, Anita; Giersig, Cecylia; Kuo, Wen-Liang; Rosner, Marsha Rich; Tang, Wei-Jen; Schmitt, Manfred

2008-11-01

Immunohistochemical evidence of ubiquitous distribution of the metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, and spleen) and on a cell microarray of 31 tumor cell lines of different origin, as well as trophoblast cells and normal blood lymphocytes and granulocytes. IDE protein was expressed in all the tissues assessed and all the tumor cell lines except for Raji and HL-60. Trophoblast cells and granulocytes, but not normal lymphocytes, were also IDE-positive.

Immunohistochemical evidence for ubiquitous distribution of metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines

PubMed Central

Weirich, Gregor; Mengele, Karin; Yfanti, Christina; Gkazepis, Apostolos; Hellmann, Daniela; Welk, Anita; Giersig, Cecylia; Kuo, Wen-Liang; Rosner, Marsha Rich; Tang, Wei-Jen; Schmitt, Manfred

2013-01-01

Immunohistochemical evidence for ubiquitous distribution of metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, spleen) and on a cell microarray encompassing 31 tumor cell lines of different origin plus trophoblast cells, and normal blood lymphocytes and granulocytes. IDE protein is expressed by all of the tissues assessed and in all of the tumor cell lines except Raji and HL-60; trophoblast cells and granulocytes but not normal lymphocytes are also IDE-positive. PMID:18783335

Expression profiling of peroxisome proliferator-activated receptor-delta (PPAR-delta) in mouse tissues using tissue microarray.

PubMed

Higashiyama, Hiroyuki; Billin, Andrew N; Okamoto, Yuji; Kinoshita, Mine; Asano, Satoshi

2007-05-01

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

The importance of prenatal 3-dimensional sonography in a case of a segmental overgrowth syndrome with unclear chromosomal microarray results.

PubMed

Asoglu, Mehmet Resit; Higgs, Amanda; Esin, Sertac; Kaplan, Julie; Turan, Sifa

2018-06-01

PIK3CA-related overgrowth spectrum, caused by mosaic mutations in the PIK3CA gene, is associated with regional or generalized asymmetric overgrowth of the body or a body part in addition to other clinical findings. Three-dimensional ultrasonography (3-D US) has the capability to display structural abnormalities in soft tissues or other organs, thereby facilitating identification of segmental overgrowth lesions. We present a case suspected of having a segmental overgrowth disorder based on 3-D US, whose chromosomal microarray result was abnormal, but apparently was not the cause of the majority of the fetus's clinical features. © 2017 Wiley Periodicals, Inc.

Derivation of Tissue-specific Functional Gene Sets to Aid Transcriptomic Analysis of Chemical Impacts on the Teleost Reproductive Axis.

EPA Science Inventory

Oligonucleotide microarrays are a powerful tool for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the poor power of microarray-based analyses to detect diffe...

Microarrays for Undergraduate Classes

ERIC Educational Resources Information Center

Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

2006-01-01

A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

PubMed Central

Kim, Min-Sik; Kuppireddy, Sarada V.; Sakamuri, Sruthi; Singal, Mukul; Getnet, Derese; Harsha, H. C.; Goel, Renu; Balakrishnan, Lavanya; Jacob, Harrys K. C.; Kashyap, Manoj K.; Tankala, Shantal G.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Jaffee, Elizabeth; Goggins, Michael G.; Velculescu, Victor E.; Hruban, Ralph H.; Pandey, Akhilesh

2013-01-01

Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research. PMID:22985314

Therapeutic potential of Dickkopf-1 in wild-type BRAF papillary thyroid cancer via regulation of β-catenin/E-cadherin signaling.

PubMed

Cho, Sun Wook; Kim, Young A; Sun, Hyun Jin; Ahn, Hwa Young; Lee, Eun Kyung; Yi, Ka Hee; Oh, Byung-Chul; Park, Do Joon; Cho, Bo Youn; Park, Young Joo

2014-09-01

Aberrant activation of the Wnt/β-catenin pathway is a common pathogenesis of various human cancers. We investigated the role of the Wnt inhibitor, Dkk-1, in papillary thyroid cancer (PTC). Immunohistochemical β-catenin staining was performed in tissue microarray containing 148 PTCs and five normal thyroid tissues. In vivo effects of Dkk-1 were explored using ectopic tumors with BHP10-3SC cells. In 27 PTC patients, 60% of patients showed β-catenin up-regulation and Dkk-1 down-regulation in tumor vs normal tissues. Tissue microarray analysis showed that 14 of 148 PTC samples exhibited cytoplasmic-dominant β-catenin expression compared to membranous-dominant expression in normal tissues. Aberrant β-catenin expression was significantly correlated with higher rates of the loss of membranous E-cadherin expression and poor disease-free survival than that in the normal membranous expression group over a median follow-up period of 14 years. Implantation of Dkk-1-overexpressing BHP10-3SC cells revealed delayed tumor growth, resulting from the rescue of membranous β-catenin and E-cadherin expressions. Furthermore, tissue microarray analysis demonstrated that BRAF(WT) patients had higher rates of aberrant expressions of β-catenin and E-cadherin than BRAF(V600E) patients. Indeed, the inhibitory effects of Dkk-1 on cell survival were more sensitive in BRAF(WT) (BHP10-3SC and TPC-1) than in BRAF(V600E) (SNU-790 and BCPAP) cells. Overexpression of BRAF(V600E) in normal thyroid epithelial (H tori) cells also reduced the effects of Dkk-1 on cell survival. A subset of PTC patients showed aberrant expression of β-catenin/E-cadherin signaling and poor disease-free survival. Dkk-1 might have a therapeutic role, particularly in BRAF(WT) patients.

TMA Navigator: network inference, patient stratification and survival analysis with tissue microarray data

PubMed Central

Lubbock, Alexander L. R.; Katz, Elad; Harrison, David J.; Overton, Ian M.

2013-01-01

Tissue microarrays (TMAs) allow multiplexed analysis of tissue samples and are frequently used to estimate biomarker protein expression in tumour biopsies. TMA Navigator (www.tmanavigator.org) is an open access web application for analysis of TMA data and related information, accommodating categorical, semi-continuous and continuous expression scores. Non-biological variation, or batch effects, can hinder data analysis and may be mitigated using the ComBat algorithm, which is incorporated with enhancements for automated application to TMA data. Unsupervised grouping of samples (patients) is provided according to Gaussian mixture modelling of marker scores, with cardinality selected by Bayesian information criterion regularization. Kaplan–Meier survival analysis is available, including comparison of groups identified by mixture modelling using the Mantel-Cox log-rank test. TMA Navigator also supports network inference approaches useful for TMA datasets, which often constitute comparatively few markers. Tissue and cell-type specific networks derived from TMA expression data offer insights into the molecular logic underlying pathophenotypes, towards more effective and personalized medicine. Output is interactive, and results may be exported for use with external programs. Private anonymous access is available, and user accounts may be generated for easier data management. PMID:23761446

Gene Expression Profiling of Gastric Cancer

PubMed Central

Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

2015-01-01

Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

Biomarkers of the Hedgehog/Smoothened pathway in healthy volunteers

PubMed Central

Kadam, Sunil K; Patel, Bharvin K R; Jones, Emma; Nguyen, Tuan S; Verma, Lalit K; Landschulz, Katherine T; Stepaniants, Sergey; Li, Bin; Brandt, John T; Brail, Leslie H

2012-01-01

The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway. PMID:22611475

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Predicting chemical bioavailability using microarray gene expression data and regression modeling: A tale of three explosive compounds.

PubMed

Gong, Ping; Nan, Xiaofei; Barker, Natalie D; Boyd, Robert E; Chen, Yixin; Wilkins, Dawn E; Johnson, David R; Suedel, Burton C; Perkins, Edward J

2016-03-08

Chemical bioavailability is an important dose metric in environmental risk assessment. Although many approaches have been used to evaluate bioavailability, not a single approach is free from limitations. Previously, we developed a new genomics-based approach that integrated microarray technology and regression modeling for predicting bioavailability (tissue residue) of explosives compounds in exposed earthworms. In the present study, we further compared 18 different regression models and performed variable selection simultaneously with parameter estimation. This refined approach was applied to both previously collected and newly acquired earthworm microarray gene expression datasets for three explosive compounds. Our results demonstrate that a prediction accuracy of R(2) = 0.71-0.82 was achievable at a relatively low model complexity with as few as 3-10 predictor genes per model. These results are much more encouraging than our previous ones. This study has demonstrated that our approach is promising for bioavailability measurement, which warrants further studies of mixed contamination scenarios in field settings.

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

PubMed Central

Galfalvy, Hanga C; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Pavlidis, Paul; Ellis, Steven P; Mann, J John; Sibille, Etienne; Arango, Victoria

2003-01-01

Background Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Results Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. Conclusion In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects. PMID:12962547

The Porcelain Crab Transcriptome and PCAD, the Porcelain Crab Microarray and Sequence Database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tagmount, Abderrahmane; Wang, Mei; Lindquist, Erika

2010-01-27

Background: With the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes. Methodology/Principal Findings: A set of ~;;30K unique sequences (UniSeqs) representing ~;;19K clusters were generated from ~;;98K high quality ESTs from a set ofmore » tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66percent of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases.Conclusions/Significance: The EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in EST library sequencing approaches, and thus represent a rich resource for studies of environmental genomics.« less

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

PubMed Central

Ramirez, Lisa S.; Wang, Jun

2016-01-01

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

Paraffin tissue microarrays constructed with a cutting board and cutting board arrayer.

PubMed

Vogel, Ulrich Felix

2010-05-01

Paraffin tissue microarrays (PTMAs) are blocks of paraffin containing up to 1300 paraffin tissue core biopsies (PTCBs). Normally, these PTCBs are punched from routine paraffin tissue blocks, which contain tissues of differing thicknesses. Therefore, the PTCBs are of different lengths. In consequence, the sections of the deeper portions of the PTMA do not contain all of the desired PTCBs. To overcome this drawback, cutting boards were constructed from panels of plastic with a thickness of 4 mm. Holes were drilled into the plastic and filled completely with at least one PTCB per hole. After being trimmed to a uniform length of 4 mm, these PTCBs were pushed from the cutting board into corresponding holes in a recipient block by means of a plate with steel pins. Up to 1000 sections per PTMA were cut without any significant loss of PTCBs, thereby increasing the efficacy of the PTMA technique.

A preliminary result of three-dimensional microarray technology to gene analysis with endoscopic ultrasound-guided fine-needle aspiration specimens and pancreatic juices

PubMed Central

2010-01-01

Background Analysis of gene expression and gene mutation may add information to be different from ordinary pathological tissue diagnosis. Since samples obtained endoscopically are very small, it is desired that more sensitive technology is developed for gene analysis. We investigated whether gene expression and gene mutation analysis by newly developed ultra-sensitive three-dimensional (3D) microarray is possible using small amount samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens and pancreatic juices. Methods Small amount samples from 17 EUS-FNA specimens and 16 pancreatic juices were obtained. After nucleic acid extraction, the samples were amplified with labeling and analyzed by the 3D microarray. Results The analyzable rate with the microarray was 46% (6/13) in EUS-FNA specimens of RNAlater® storage, and RNA degradations were observed in all the samples of frozen storage. In pancreatic juices, the analyzable rate was 67% (4/6) in frozen storage samples and 20% (2/10) in RNAlater® storage. EUS-FNA specimens were classified into cancer and non-cancer by gene expression analysis and K-ras codon 12 mutations were also detected using the 3D microarray. Conclusions Gene analysis from small amount samples obtained endoscopically was possible by newly developed 3D microarray technology. High quality RNA from EUS-FNA samples were obtained and remained in good condition only using RNA stabilizer. In contrast, high quality RNA from pancreatic juice samples were obtained only in frozen storage without RNA stabilizer. PMID:20416107

A Graphical Systems Model and Tissue-specific Functional Gene Sets to Aid Transcriptomic Analysis of Chemical Impacts on the Female Teleost Reproductive Axis

EPA Science Inventory

Oligonucleotide microarrays and other ‘omics’ approaches are powerful tools for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the poor power of microarray-b...

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

PubMed

Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

2016-01-01

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Comparison of gene expression profiles between dental pulp and periodontal ligament tissues in humans

PubMed Central

Gong, Ai-Xiu; Zhang, Jing-Han; Li, Jing; Wu, Jun; Wang, Lin; Miao, Deng-Shun

2017-01-01

There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration. PMID:28713908

Implementation of plaid model biclustering method on microarray of carcinoma and adenoma tumor gene expression data

NASA Astrophysics Data System (ADS)

Ardaneswari, Gianinna; Bustamam, Alhadi; Sarwinda, Devvi

2017-10-01

A Tumor is an abnormal growth of cells that serves no purpose. Carcinoma is a tumor that grows from the top of the cell membrane and the organ adenoma is a benign tumor of the gland-like cells or epithelial tissue. In the field of molecular biology, the development of microarray technology is used in the data store of disease genetic expression. For each of microarray gene, an amount of information is stored for each trait or condition. In gene expression data clustering can be done with a bicluster algorithm, thats clustering method which not only the objects to be clustered, but also the properties or condition of the object. This research proposed Plaid Model Biclustering as one of biclustering method. In this study, we discuss the implementation of Plaid Model Biclustering Method on microarray of Carcinoma and Adenoma tumor gene expression data. From the experimental results, we found three biclusters are formed by Carcinoma gene expression data and four biclusters are formed by Adenoma gene expression data.

Comparison of Dorsocervical With Abdominal Subcutaneous Adipose Tissue in Patients With and Without Antiretroviral Therapy–Associated Lipodystrophy

PubMed Central

Sevastianova, Ksenia; Sutinen, Jussi; Greco, Dario; Sievers, Meline; Salmenkivi, Kaisa; Perttilä, Julia; Olkkonen, Vesa M.; Wågsäter, Dick; Lidell, Martin E.; Enerbäck, Sven; Eriksson, Per; Walker, Ulrich A.; Auvinen, Petri; Ristola, Matti; Yki-Järvinen, Hannele

2011-01-01

OBJECTIVE Combination antiretroviral therapy (cART) is associated with lipodystrophy, i.e., loss of subcutaneous adipose tissue in the abdomen, limbs, and face and its accumulation intra-abdominally. No fat is lost dorsocervically and it can even accumulate in this region (buffalo hump). It is unknown how preserved dorsocervical fat differs from abdominal subcutaneous fat in HIV-1–infected cART-treated patients with (cART+LD+) and without (cART+LD−) lipodystrophy. RESEARCH DESIGN AND METHODS We used histology, microarray, PCR, and magnetic resonance imaging to compare dorsocervical and abdominal subcutaneous adipose tissue in cART+LD+ (n = 21) and cART+LD− (n = 11). RESULTS Albeit dorsocervical adipose tissue in cART+LD+ seems spared from lipoatrophy, its mitochondrial DNA (mtDNA; copies/cell) content was significantly lower (by 62%) than that of the corresponding tissue in cART+LD−. Expression of CD68 mRNA, a marker of macrophages, and numerous inflammatory genes in microarray were significantly lower in dorsocervical versus abdominal subcutaneous adipose tissue. Genes with the greatest difference in expression between the two depots were those involved in regulation of transcription and regionalization (homeobox genes), irrespective of lipodystrophy status. There was negligible mRNA expression of uncoupling protein 1, a gene characteristic of brown adipose tissue, in either depot. CONCLUSIONS Because mtDNA is depleted even in the nonatrophic dorsocervical adipose tissue, it is unlikely that the cause of lipoatrophy is loss of mtDNA. Dorsocervical adipose tissue is less inflamed than lipoatrophic adipose tissue. It does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal subcutaneous adipose tissue is in expression of homeobox genes. PMID:21602514

Vitamin D Receptor gene (VDR) transcripts in bone, cartilage, muscles and blood and microarray analysis of vitamin D responsive genes expression in paravertebral muscles of Juvenile and Adolescent Idiopathic Scoliosis patients

PubMed Central

2012-01-01

Background VDR may be considered as a candidate gene potentially related to Idiopathic Scoliosis susceptibility and natural history. Transcriptional profile of VDR mRNA isoforms might be changed in the structural tissues of the scoliotic spine and potentially influence the expression of VDR responsive genes. The purpose of the study was to determine differences in mRNA abundance of VDR isoforms in bone, cartilage and paravertebral muscles between tissues from curve concavity and convexity, between JIS and AIS and to identify VDR responsive genes differentiating Juvenile and Adolescent Idiopathic Scoliosis in paravertebral muscles. Methods In a group of 29 patients with JIS and AIS, specimens of bone, cartilage, paravertebral muscles were harvested at the both sides of the curve apex together with peripheral blood samples. Extracted total RNA served as a matrix for VDRs and VDRl mRNA quantification by QRT PCR. Subsequent microarray analysis of paravertebral muscular tissue samples was performed with HG U133A chips (Affymetrix). Quantitative data were compared by a nonparametric Mann Whitney U test. Microarray results were analyzed with GeneSpring 11GX application. Matrix plot of normalized log-intensities visualized the degree of differentiation between muscular tissue transcriptomes of JIS and AIS group. Fold Change Analysis with cutoff of Fold Change ≥2 identified differentially expressed VDR responsive genes in paravertebral muscles of JIS and AIS. Results No significant differences in transcript abundance of VDR isoforms between tissues of the curve concavity and convexity were found. Statistically significant difference between JIS and AIS group in mRNA abundance of VDRl isoform was found in paravertebral muscles of curve concavity. Higher degree of muscular transcriptome differentiation between curve concavity and convexity was visualized in JIS group. In paravertebral muscles Tob2 and MED13 were selected as genes differentially expressed in JIS and AIS group. Conclusions In Idiopathic Scolioses transcriptional activity and alternative splicing of VDR mRNA in osseous, cartilaginous, and paravertebral muscular tissues are tissue specific and equal on both sides of the curve. The number of mRNA copies of VDRl izoform in concave paravertebral muscles might be one of the factors differentiating JIS and AIS. In paravertebral muscles Tob2 and Med13 genes differentiate Adolescent and Juvenile type of Idiopathic Scoliosis. PMID:23259508

Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments.

PubMed

Akkiprik, Mustafa; Peker, İrem; Özmen, Tolga; Amuran, Gökçe Güllü; Güllüoğlu, Bahadır M; Kaya, Handan; Özer, Ayşe

2015-11-10

IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer.

A novel surface modification approach for protein and cell microarrays

NASA Astrophysics Data System (ADS)

Kurkuri, Mahaveer D.; Driever, Chantelle; Thissen, Helmut W.; Voelcker, Nicholas H.

2007-01-01

Tissue engineering and stem cell technologies have led to a rapidly increasing interest in the control of the behavior of mammalian cells growing on tissue culture substrates. Multifunctional polymer coatings can assist research in this area in many ways, for example, by providing low non-specific protein adsorption properties and reactive functional groups at the surface. The latter can be used for immobilization of specific biological factors that influence cell behavior. In this study, glass slides were coated with copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). The coatings were prepared by three different methods based on dip and spin coating as well as polymer grafting procedures. Coatings were characterized by X-ray photoelectron spectroscopy, surface sensitive infrared spectroscopy, ellipsometry and contact angle measurements. A fluorescently labelled protein was deposited onto reactive coatings using a contact microarrayer. Printing of a model protein (fluorescein labeled bovine serum albumin) was performed at different protein concentrations, pH, temperature, humidity and using different micropins. The arraying of proteins was studied with a microarray scanner. Arrays printed at a protein concentration above 50 μg/mL prepared in pH 5 phosphate buffer at 10°C and 65% relative humidity gave the most favourable results in terms of the homogeneity of the printed spots and the fluorescence intensity.

MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

PubMed

Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

2009-05-01

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles.

PubMed

Bălăcescu, Loredana; Bălăcescu, O; Crişan, N; Fetica, B; Petruţ, B; Bungărdean, Cătălina; Rus, Meda; Tudoran, Oana; Meurice, G; Irimie, Al; Dragoş, N; Berindan-Neagoe, Ioana

2011-01-01

Prostate cancer represents the first leading cause of cancer among western male population, with different clinical behavior ranging from indolent to metastatic disease. Although many molecules and deregulated pathways are known, the molecular mechanisms involved in the development of prostate cancer are not fully understood. The aim of this study was to explore the molecular variation underlying the prostate cancer, based on microarray analysis and bioinformatics approaches. Normal and prostate cancer tissues were collected by macrodissection from prostatectomy pieces. All prostate cancer specimens used in our study were Gleason score 7. Gene expression microarray (Agilent Technologies) was used for Whole Human Genome evaluation. The bioinformatics and functional analysis were based on Limma and Ingenuity software. The microarray analysis identified 1119 differentially expressed genes between prostate cancer and normal prostate, which were up- or down-regulated at least 2-fold. P-values were adjusted for multiple testing using Benjamini-Hochberg method with a false discovery rate of 0.01. These genes were analyzed with Ingenuity Pathway Analysis software and were established 23 genetic networks. Our microarray results provide new information regarding the molecular networks in prostate cancer stratified as Gleason 7. These data highlighted gene expression profiles for better understanding of prostate cancer progression.

Microarray-based identification of differentially expressed genes in extramammary Paget’s disease

PubMed Central

Lin, Jin-Ran; Liang, Jun; Zhang, Qiao-An; Huang, Qiong; Wang, Shang-Shang; Qin, Hai-Hong; Chen, Lian-Jun; Xu, Jin-Hua

2015-01-01

Extramammary Paget’s disease (EMPD) is a rare cutaneous malignancy accounting for approximately 1-2% of vulvar cancers. The rarity of this disease has caused difficulties in characterization and the molecular mechanism underlying EMPD development remains largely unclear. Here we used microarray analysis to identify differentially expressed genes in EMPD of the scrotum comparing with normal epithelium from healthy donors. Agilent single-channel microarray was used to compare the gene expression between 6 EMPD specimens and 6 normal scrotum epithelium samples. A total of 799 up-regulated genes and 723 down-regulated genes were identified in EMPD tissues. Real-time PCR was conducted to verify the differential expression of some representative genes, including ERBB4, TCF3, PAPSS2, PIK3R3, PRLR, SULT1A1, TCF7L1, and CREB3L4. Generally, the real-time PCR results were consistent with microarray data, and the expression of ERBB4, PRLR, TCF3, PIK3R3, SULT1A1, and TCF7L1 was significantly overexpressed in EMPD (P<0.05). Moreover, the overexpression of PRLR in EMPD, a receptor for the anterior pituitary hormone prolactin (PRL), was confirmed by immunohistochemistry. These data demonstrate that the differentially expressed genes from the microarray-based identification are tightly associated with EMPD occurrence. PMID:26221264

Symptomatic and asymptomatic benign prostatic hyperplasia: Molecular differentiation by using microarrays

NASA Astrophysics Data System (ADS)

Prakash, Kulkarni; Pirozzi, Gregorio; Elashoff, Michael; Munger, William; Waga, Iwao; Dhir, Rajiv; Kakehi, Yoshiyuki; Getzenberg, Robert H.

2002-05-01

Benign prostatic hyperplasia (BPH) is a disease of unknown etiology that significantly affects the quality of life in aging men. Histologic BPH may present itself either as symptomatic or asymptomatic in nature. To elucidate the molecular differences underlying BPH, gene expression profiles from the prostate transition zone tissue have been analyzed by using microarrays. A set of 511 differentially expressed genes distinguished symptomatic and asymptomatic BPH. This genetic signature separates BPH from normal tissue but does not seem to change with age. These data could provide novel approaches for alleviating symptoms and hyperplasia in BPH.

Relevance of TNBS-Colitis in Rats: A Methodological Study with Endoscopic, Histologic and Transcriptomic Characterization and Correlation to IBD

PubMed Central

Brenna, Øystein; Furnes, Marianne W.; Drozdov, Ignat; van Beelen Granlund, Atle; Flatberg, Arnar; Sandvik, Arne K.; Zwiggelaar, Rosalie T. M.; Mårvik, Ronald; Nordrum, Ivar S.; Kidd, Mark; Gustafsson, Björn I.

2013-01-01

Background Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD. Methodology/Principal Findings Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1α, IL-1β, TLR2, TLR4, PRNP were all significantly up-regulated, while PPARγ was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1α. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing. Conclusions/Significance Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD. PMID:23382912

Overexpression of Plasminogen Activator Inhibitor-1 in Advanced Gastric Cancer with Aggressive Lymph Node Metastasis

PubMed Central

Suh, Yun-Suhk; Yu, Jieun; Kim, Byung Chul; Choi, Boram; Han, Tae-Su; Ahn, Hye Seong; Kong, Seong-Ho; Lee, Hyuk-Joon; Kim, Woo Ho; Yang, Han-Kwang

2015-01-01

Purpose The purpose of this study is to investigate differentially expressed genes using DNA microarray between advanced gastric cancer (AGC) with aggressive lymph node (LN) metastasis and that with a more advanced tumor stage but without LN metastasis. Materials and Methods Five sample pairs of gastric cancer tissue and normal gastric mucosa were taken from three patients with T3N3 stage (highN) and two with T4N0 stage (lowN). Data from triplicate DNA microarray experiments were analyzed, and candidate genes were identified using a volcano plot that showed ≥ 2-fold differential expression and were significant by Welch's t test (p < 0.05) between highN and lowN. Those selected genes were validated independently by reverse-transcriptase–polymerase chain reaction (RT-PCR) using five AGC patients, and tissue-microarray (TMA) comprising 47 AGC patients. Results CFTR, LAMC2, SERPINE2, F2R, MMP7, FN1, TIMP1, plasminogen activator inhibitor-1 (PAI-1), ITGB8, SDS, and TMPRSS4 were commonly up-regulated over 2-fold in highN. REG3A, CD24, ITLN1, and WBP5 were commonly down-regulated over 2-fold in lowN. Among these genes, overexpression of PAI-1 was validated by RT-PCR, and TMA showed 16.7% (7/42) PAI-1 expression in T3N3, but none (0/5) in T4N0 (p=0.393). Conclusion DNA microarray analysis and validation by RT-PCR and TMA showed that overexpression of PAI-1 is related to aggressive LN metastasis in AGC. PMID:25687870

Tissue microarrays and quantitative tissue-based image analysis as a tool for oncology biomarker and diagnostic development.

PubMed

Dolled-Filhart, Marisa P; Gustavson, Mark D

2012-11-01

Translational oncology has been improved by using tissue microarrays (TMAs), which facilitate biomarker analysis of large cohorts on a single slide. This has allowed for rapid analysis and validation of potential biomarkers for prognostic and predictive value, as well as for evaluation of biomarker prevalence. Coupled with quantitative analysis of immunohistochemical (IHC) staining, objective and standardized biomarker data from tumor samples can further advance companion diagnostic approaches for the identification of drug-responsive or resistant patient subpopulations. This review covers the advantages, disadvantages and applications of TMAs for biomarker research. Research literature and reviews of TMAs and quantitative image analysis methodology have been surveyed for this review (with an AQUA® analysis focus). Applications such as multi-marker diagnostic development and pathway-based biomarker subpopulation analyses are described. Tissue microarrays are a useful tool for biomarker analyses including prevalence surveys, disease progression assessment and addressing potential prognostic or predictive value. By combining quantitative image analysis with TMAs, analyses will be more objective and reproducible, allowing for more robust IHC-based diagnostic test development. Quantitative multi-biomarker IHC diagnostic tests that can predict drug response will allow for greater success of clinical trials for targeted therapies and provide more personalized clinical decision making.

HOXB9 Expression Correlates with Histological Grade and Prognosis in LSCC

PubMed Central

2017-01-01

The purpose of this study was to investigate the HOX gene expression profile in laryngeal squamous cell carcinoma (LSCC) and assess whether some genes are associated with the clinicopathological features and prognosis in LSCC patients. The HOX gene levels were tested by microarray and validated by qRT-PCR in paired cancerous and adjacent noncancerous LSCC tissue samples. The microarray testing data of 39 HOX genes revealed 15 HOX genes that were at least 2-fold upregulated and 2 that were downregulated. After qRT-PCR evaluation, the three most upregulated genes (HOXB9, HOXB13, and HOXD13) were selected for tissue microarray (TMA) analysis. The correlations between the HOXB9, HOXB13, and HOXD13 expression levels and both clinicopathological features and prognosis were analyzed. Three HOX gene expression levels were markedly increased in LSCC tissues compared with adjacent noncancerous tissues (P < 0.001). HOXB9 was found to correlate with histological grade (P < 0.01) and prognosis (P < 0.01) in LSCC. In conclusion, this study revealed that HOXB9, HOXB13, and HOXD13 were upregulated and may play important roles in LSCC. Moreover, HOXB9 may serve as a novel marker of poor prognosis and a potential therapeutic target in LSCC patients. PMID:28808656

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

PubMed

Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

2003-01-01

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Microarray Data Processing Techniques for Genome-Scale Network Inference from Large Public Repositories.

PubMed

Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

2016-09-19

Pre-processing of microarray data is a well-studied problem. Furthermore, all popular platforms come with their own recommended best practices for differential analysis of genes. However, for genome-scale network inference using microarray data collected from large public repositories, these methods filter out a considerable number of genes. This is primarily due to the effects of aggregating a diverse array of experiments with different technical and biological scenarios. Here we introduce a pre-processing pipeline suitable for inferring genome-scale gene networks from large microarray datasets. We show that partitioning of the available microarray datasets according to biological relevance into tissue- and process-specific categories significantly extends the limits of downstream network construction. We demonstrate the effectiveness of our pre-processing pipeline by inferring genome-scale networks for the model plant Arabidopsis thaliana using two different construction methods and a collection of 11,760 Affymetrix ATH1 microarray chips. Our pre-processing pipeline and the datasets used in this paper are made available at http://alurulab.cc.gatech.edu/microarray-pp.

A comprehensive transcript index of the human genome generated using microarrays and computational approaches

PubMed Central

Schadt, Eric E; Edwards, Stephen W; GuhaThakurta, Debraj; Holder, Dan; Ying, Lisa; Svetnik, Vladimir; Leonardson, Amy; Hart, Kyle W; Russell, Archie; Li, Guoya; Cavet, Guy; Castle, John; McDonagh, Paul; Kan, Zhengyan; Chen, Ronghua; Kasarskis, Andrew; Margarint, Mihai; Caceres, Ramon M; Johnson, Jason M; Armour, Christopher D; Garrett-Engele, Philip W; Tsinoremas, Nicholas F; Shoemaker, Daniel D

2004-01-01

Background Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22. Results The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale. Conclusions These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized. PMID:15461792

Successful Application of Microarray Technology to Microdissected Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

Coudry, Renata A.; Meireles, Sibele I.; Stoyanova, Radka; Cooper, Harry S.; Carpino, Alan; Wang, Xianqun; Engstrom, Paul F.; Clapper, Margie L.

2007-01-01

The establishment of a reliable method for using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue would provide an opportunity to obtain novel gene expression data from the vast amounts of archived tissue. A custom-designed 22,000 oligonucleotide array was used in the present study to compare the gene expression profile of colonic epithelial cells isolated by laser capture microdissection from FFPE-archived samples with that of the same cell population from matched frozen samples, the preferred source of RNA. Total RNA was extracted from FFPE tissues, amplified, and labeled using the Paradise Reagent System. The quality of the input RNA was assessed by the Bioanalyzer profile, reverse transcriptase-polymerase chain reaction, and agarose gel electrophoresis. The results demonstrate that it is possible to obtain reliable microarray data from FFPE samples using RNA acquired by laser capture microdissection. The concordance between matched FFPE and frozen samples was evaluated and expressed as a Pearson’s correlation coefficient, with values ranging from 0.80 to 0.97. The presence of ribosomal RNA peaks in FFPE-derived RNA was reflected by a high correlation with paired frozen samples. A set of practical recommendations for evaluating the RNA integrity and quality in FFPE samples is reported. PMID:17251338

The Prostate Cancer Biorepository Network (PCBN)

DTIC Science & Technology

2016-10-01

site includes blood (serum, plasma, and buffy coat), prostatectomy tissues (frozen), biopsies and metastatic tissue from rapid autopsies (paraffin...embedded material and tissue microarrays (TMAs)), prostate cancer patient derived xenografts (PDX) and derived specimens (DNA and RNA) from prostate...Genitourinary Cancer Biorepository set up a rapid autopsy program to provide access to metastatic tissue and create patient derived xenograft (PDX

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

PubMed

Galfalvy, Hanga C; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Pavlidis, Paul; Ellis, Steven P; Mann, J John; Sibille, Etienne; Arango, Victoria

2003-09-08

Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects.

Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

PubMed

Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C

2016-04-30

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

Altered Molecular Expression of the TLR4/NF-κB Signaling Pathway in Mammary Tissue of Chinese Holstein Cattle with Mastitis

PubMed Central

Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

2015-01-01

Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis. PMID:25706977

Altered molecular expression of the TLR4/NF-κB signaling pathway in mammary tissue of Chinese Holstein cattle with mastitis.

PubMed

Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

2015-01-01

Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis.

Cancer diagnosis marker extraction for soft tissue sarcomas based on gene expression profiling data by using projective adaptive resonance theory (PART) filtering method

PubMed Central

Takahashi, Hiro; Nemoto, Takeshi; Yoshida, Teruhiko; Honda, Hiroyuki; Hasegawa, Tadashi

2006-01-01

Background Recent advances in genome technologies have provided an excellent opportunity to determine the complete biological characteristics of neoplastic tissues, resulting in improved diagnosis and selection of treatment. To accomplish this objective, it is important to establish a sophisticated algorithm that can deal with large quantities of data such as gene expression profiles obtained by DNA microarray analysis. Results Previously, we developed the projective adaptive resonance theory (PART) filtering method as a gene filtering method. This is one of the clustering methods that can select specific genes for each subtype. In this study, we applied the PART filtering method to analyze microarray data that were obtained from soft tissue sarcoma (STS) patients for the extraction of subtype-specific genes. The performance of the filtering method was evaluated by comparison with other widely used methods, such as signal-to-noise, significance analysis of microarrays, and nearest shrunken centroids. In addition, various combinations of filtering and modeling methods were used to extract essential subtype-specific genes. The combination of the PART filtering method and boosting – the PART-BFCS method – showed the highest accuracy. Seven genes among the 15 genes that are frequently selected by this method – MIF, CYFIP2, HSPCB, TIMP3, LDHA, ABR, and RGS3 – are known prognostic marker genes for other tumors. These genes are candidate marker genes for the diagnosis of STS. Correlation analysis was performed to extract marker genes that were not selected by PART-BFCS. Sixteen genes among those extracted are also known prognostic marker genes for other tumors, and they could be candidate marker genes for the diagnosis of STS. Conclusion The procedure that consisted of two steps, such as the PART-BFCS and the correlation analysis, was proposed. The results suggest that novel diagnostic and therapeutic targets for STS can be extracted by a procedure that includes the PART filtering method. PMID:16948864

Methodological Challenges in Protein Microarray and Immunohistochemistry for the Discovery of Novel Autoantibodies in Paediatric Acute Disseminated Encephalomyelitis

PubMed Central

Peschl, Patrick; Ramberger, Melanie; Höftberger, Romana; Jöhrer, Karin; Baumann, Matthias; Rostásy, Kevin; Reindl, Markus

2017-01-01

Acute disseminated encephalomyelitis (ADEM) is a rare autoimmune-mediated demyelinating disease affecting mainly children and young adults. Differentiation to multiple sclerosis is not always possible, due to overlapping clinical symptoms and recurrent and multiphasic forms. Until now, immunoglobulins reactive to myelin oligodendrocyte glycoprotein (MOG antibodies) have been found in a subset of patients with ADEM. However, there are still patients lacking autoantibodies, necessitating the identification of new autoantibodies as biomarkers in those patients. Therefore, we aimed to identify novel autoantibody targets in ADEM patients. Sixteen ADEM patients (11 seronegative, 5 seropositive for MOG antibodies) were analysed for potential new biomarkers, using a protein microarray and immunohistochemistry on rat brain tissue to identify antibodies against intracellular and surface neuronal and glial antigens. Nine candidate antigens were identified in the protein microarray analysis in at least two patients per group. Immunohistochemistry on rat brain tissue did not reveal new target antigens. Although no new autoantibody targets could be found here, future studies should aim to identify new biomarkers for therapeutic and prognostic purposes. The microarray analysis and immunohistochemistry methods used here have several limitations, which should be considered in future searches for biomarkers. PMID:28327523

Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis

PubMed Central

Abbott, Karen L.; Lim, Jae-Min; Wells, Lance; Benigno, Benedict B.; McDonald, John F.; Pierce, Michael

2016-01-01

Epithelial ovarian cancer is diagnosed less than 25% of the time when the cancer is confined to the ovary, leading to 5-year survival rates of less than 30%. Therefore, there is an urgent need for early diagnostics for ovarian cancer. Our study using glycotranscriptome comparative analysis of endometrioid ovarian cancer tissue and normal ovarian tissue led to the identification of distinct differences in the transcripts of a restricted set of glycosyltransferases involved in N-linked glycosylation. Utilizing lectins that bind to glycan structures predicted to show changes, we observed differences in lectin-bound glycoproteins consistent with some of the transcript differences. In this study, we have extended our observations by the use of selected lectins to perform a targeted glycoproteomic analysis of ovarian cancer and normal ovarian tissues. Our results have identified several glycoproteins that display tumor-specific glycosylation changes. We have verified these glycosylation changes on glycoproteins from tissue using immunoprecipitation followed by lectin blot detection. The glycoproteins that were verified were then analyzed further using existing microarray data obtained from benign ovarian adenomas, borderline ovarian adenocarcinomas, and malignant ovarian adenocarcinomas. The verified glycoproteins found to be expressed above control levels in the microarray data sets were then screened for tumor-specific glycan modifications in serum from ovarian cancer patients. Results obtained from two of these glycoprotein markers, periostin and thrombospondin, have confirmed that tumor-specific glycan changes can be used to distinguish ovarian cancer patient serum from normal serum. PMID:19953551

Expression of BMI-1 and Mel-18 in breast tissue - a diagnostic marker in patients with breast cancer

PubMed Central

2010-01-01

Background Polycomb Group (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer. Expression of Mel-18 and Bmi-1 has been studied in tumor tissue, but not in adjacent non-cancerous breast epithelium. Our study compares the expression of the two genes in normal breast epithelium of cancer patients and relates it to the level of expression in the corresponding tumors as well as in breast epithelium of healthy women. Methods A total of 79 tumors, of which 71 malignant tumors of the breast, 6 fibroadenomas, and 2 DCIS were studied and compared to the reduction mammoplastic specimens of 11 healthy women. In addition there was available adjacent cancer free tissue for 23 of the malignant tumors. The tissue samples were stored in RNAlater, RNA was isolated to create expression microarray profile. These two genes were then studied more closely first on mRNA transcription level by microarrays (Agilent 44 K) and quantitative RT-PCR (TaqMan) and then on protein expression level using immunohistochemistry. Results Bmi-1 mRNA is significantly up-regulated in adjacent normal breast tissue in breast cancer patients compared to normal breast tissue from noncancerous patients. Conversely, mRNA transcription level of Mel-18 is lower in normal breast from patients operated for breast cancer compared to breast tissue from mammoplasty. When protein expression of these two genes was evaluated, we observed that most of the epithelial cells were positive for Bmi-1 in both groups of tissue samples, although the expression intensity was stronger in normal tissue from cancer patients compared to mammoplasty tissue samples. Protein expression of Mel-18 showed inversely stronger intensity in tissue samples from mammoplasty compared to normal breast tissue from patients operated for breast cancer. Conclusion Bmi-1 mRNA level is consistently increased and Mel-18 mRNA level is consistently decreased in adjacent normal breast tissue of cancer patients as compared to normal breast tissue in women having had reduction mammoplasties. Bmi-1/Mel-18 ratio can be potentially used as a tool for stratifying women at risk of developing malignancy. PMID:21162745

The tissue microarray data exchange specification: A community-based, open source tool for sharing tissue microarray data

PubMed Central

Berman, Jules J; Edgerton, Mary E; Friedman, Bruce A

2003-01-01

Background Tissue Microarrays (TMAs) allow researchers to examine hundreds of small tissue samples on a single glass slide. The information held in a single TMA slide may easily involve Gigabytes of data. To benefit from TMA technology, the scientific community needs an open source TMA data exchange specification that will convey all of the data in a TMA experiment in a format that is understandable to both humans and computers. A data exchange specification for TMAs allows researchers to submit their data to journals and to public data repositories and to share or merge data from different laboratories. In May 2001, the Association of Pathology Informatics (API) hosted the first in a series of four workshops, co-sponsored by the National Cancer Institute, to develop an open, community-supported TMA data exchange specification. Methods A draft tissue microarray data exchange specification was developed through workshop meetings. The first workshop confirmed community support for the effort and urged the creation of an open XML-based specification. This was to evolve in steps with approval for each step coming from the stakeholders in the user community during open workshops. By the fourth workshop, held October, 2002, a set of Common Data Elements (CDEs) was established as well as a basic strategy for organizing TMA data in self-describing XML documents. Results The TMA data exchange specification is a well-formed XML document with four required sections: 1) Header, containing the specification Dublin Core identifiers, 2) Block, describing the paraffin-embedded array of tissues, 3)Slide, describing the glass slides produced from the Block, and 4) Core, containing all data related to the individual tissue samples contained in the array. Eighty CDEs, conforming to the ISO-11179 specification for data elements constitute XML tags used in the TMA data exchange specification. A set of six simple semantic rules describe the complete data exchange specification. Anyone using the data exchange specification can validate their TMA files using a software implementation written in Perl and distributed as a supplemental file with this publication. Conclusion The TMA data exchange specification is now available in a draft form with community-approved Common Data Elements and a community-approved general file format and data structure. The specification can be freely used by the scientific community. Efforts sponsored by the Association for Pathology Informatics to refine the draft TMA data exchange specification are expected to continue for at least two more years. The interested public is invited to participate in these open efforts. Information on future workshops will be posted at (API we site). PMID:12769826

Systematic gene microarray analysis of the lncRNA expression profiles in human uterine cervix carcinoma.

PubMed

Chen, Jie; Fu, Ziyi; Ji, Chenbo; Gu, Pingqing; Xu, Pengfei; Yu, Ningzhu; Kan, Yansheng; Wu, Xiaowei; Shen, Rong; Shen, Yan

2015-05-01

The human uterine cervix carcinoma is one of the most well-known malignancy reproductive system cancers, which threatens women health globally. However, the mechanisms of the oncogenesis and development process of cervix carcinoma are not yet fully understood. Long non-coding RNAs (lncRNAs) have been proved to play key roles in various biological processes, especially development of cancer. The function and mechanism of lncRNAs on cervix carcinoma is still rarely reported. We selected 3 cervix cancer and normal cervix tissues separately, then performed lncRNA microarray to detect the differentially expressed lncRNAs. Subsequently, we explored the potential function of these dysregulated lncRNAs through online bioinformatics databases. Finally, quantity real-time PCR was carried out to confirm the expression levels of these dysregulated lncRNAs in cervix cancer and normal tissues. We uncovered the profiles of differentially expressed lncRNAs between normal and cervix carcinoma tissues by using the microarray techniques, and found 1622 upregulated and 3026 downregulated lncRNAs (fold-change>2.0) in cervix carcinoma compared to the normal cervical tissue. Furthermore, we found HOXA11-AS might participate in cervix carcinogenesis by regulating HOXA11, which is involved in regulating biological processes of cervix cancer. This study afforded expression profiles of lncRNAs between cervix carcinoma tissue and normal cervical tissue, which could provide database for further research about the function and mechanism of key-lncRNAs in cervix carcinoma, and might be helpful to explore potential diagnosis factors and therapeutic targets for cervix carcinoma. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes.

PubMed

Mazzarelli, Joan M; Brestelli, John; Gorski, Regina K; Liu, Junmin; Manduchi, Elisabetta; Pinney, Deborah F; Schug, Jonathan; White, Peter; Kaestner, Klaus H; Stoeckert, Christian J

2007-01-01

EPConDB (http://www.cbil.upenn.edu/EPConDB) is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT-PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

Transcriptomic responses to wounding: meta-analysis of gene expression microarray data.

PubMed

Sass, Piotr Andrzej; Dąbrowski, Michał; Charzyńska, Agata; Sachadyn, Paweł

2017-11-07

A vast amount of microarray data on transcriptomic response to injury has been collected so far. We designed the analysis in order to identify the genes displaying significant changes in expression after wounding in different organisms and tissues. This meta-analysis is the first study to compare gene expression profiles in response to wounding in as different tissues as heart, liver, skin, bones, and spinal cord, and species, including rat, mouse and human. We collected available microarray transcriptomic profiles obtained from different tissue injury experiments and selected the genes showing a minimum twofold change in expression in response to wounding in prevailing number of experiments for each of five wound healing stages we distinguished: haemostasis & early inflammation, inflammation, early repair, late repair and remodelling. During the initial phases after wounding, haemostasis & early inflammation and inflammation, the transcriptomic responses showed little consistency between different tissues and experiments. For the later phases, wound repair and remodelling, we identified a number of genes displaying similar transcriptional responses in all examined tissues. As revealed by ontological analyses, activation of certain pathways was rather specific for selected phases of wound healing, such as e.g. responses to vitamin D pronounced during inflammation. Conversely, we observed induction of genes encoding inflammatory agents and extracellular matrix proteins in all wound healing phases. Further, we selected several genes differentially upregulated throughout different stages of wound response, including established factors of wound healing in addition to those previously unreported  in this context such as PTPRC and AQP4. We found that transcriptomic responses to wounding showed similar traits in a diverse selection of tissues including skin, muscles, internal organs and nervous system. Notably, we distinguished transcriptional induction of inflammatory genes not only in the initial response to wounding, but also later, during wound repair and tissue remodelling.

Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

PubMed

Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

2016-07-01

The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

Tumor Acquisition for Biomarker Research in Lung Cancer

PubMed Central

Stevenson, Marvaretta; Christensen, Jared; Shoemaker, Debra; Foster, Traci; Barry, William T.; Tong, Betty C.; Wahidi, Momen; Shofer, Scott; Datto, Michael; Ginsburg, Geoffrey; Crawford, Jeffrey; D’Amico, Thomas; Ready, Neal

2015-01-01

The biopsy collection data from two lung cancer trials that required fresh tumor samples be obtained for microarray analysis were reviewed. In the trial for advanced disease, microarray data were obtained on 50 patient samples, giving an overall success rate of 60.2%. The majority of the specimens were obtained through CT-guided lung biopsies (N=30). In the trial for early-stage patients, 28 tissue specimens were collected from excess tumor after surgical resection with a success rate of 85.7%. This tissue procurement program documents the feasibility in obtaining fresh tumor specimens prospectively that could be used for molecular testing. PMID:24810245

Differentiation of the seven major lyssavirus species by oligonucleotide microarray.

PubMed

Xi, Jin; Guo, Huancheng; Feng, Ye; Xu, Yunbin; Shao, Mingfu; Su, Nan; Wan, Jiayu; Li, Jiping; Tu, Changchun

2012-03-01

An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

ATMAD: robust image analysis for Automatic Tissue MicroArray De-arraying.

PubMed

Nguyen, Hoai Nam; Paveau, Vincent; Cauchois, Cyril; Kervrann, Charles

2018-04-19

Over the last two decades, an innovative technology called Tissue Microarray (TMA), which combines multi-tissue and DNA microarray concepts, has been widely used in the field of histology. It consists of a collection of several (up to 1000 or more) tissue samples that are assembled onto a single support - typically a glass slide - according to a design grid (array) layout, in order to allow multiplex analysis by treating numerous samples under identical and standardized conditions. However, during the TMA manufacturing process, the sample positions can be highly distorted from the design grid due to the imprecision when assembling tissue samples and the deformation of the embedding waxes. Consequently, these distortions may lead to severe errors of (histological) assay results when the sample identities are mismatched between the design and its manufactured output. The development of a robust method for de-arraying TMA, which localizes and matches TMA samples with their design grid, is therefore crucial to overcome the bottleneck of this prominent technology. In this paper, we propose an Automatic, fast and robust TMA De-arraying (ATMAD) approach dedicated to images acquired with brightfield and fluorescence microscopes (or scanners). First, tissue samples are localized in the large image by applying a locally adaptive thresholding on the isotropic wavelet transform of the input TMA image. To reduce false detections, a parametric shape model is considered for segmenting ellipse-shaped objects at each detected position. Segmented objects that do not meet the size and the roundness criteria are discarded from the list of tissue samples before being matched with the design grid. Sample matching is performed by estimating the TMA grid deformation under the thin-plate model. Finally, thanks to the estimated deformation, the true tissue samples that were preliminary rejected in the early image processing step are recognized by running a second segmentation step. We developed a novel de-arraying approach for TMA analysis. By combining wavelet-based detection, active contour segmentation, and thin-plate spline interpolation, our approach is able to handle TMA images with high dynamic, poor signal-to-noise ratio, complex background and non-linear deformation of TMA grid. In addition, the deformation estimation produces quantitative information to asset the manufacturing quality of TMAs.

Oligonucleotide microarray analysis reveals dysregulation of energy-related metabolism in insulin-sensitive tissues of type 2 diabetes patients.

PubMed

Wang, M; Wang, X C; Zhao, L; Zhang, Y; Yao, L L; Lin, Y; Peng, Y D; Hu, R M

2014-06-17

Impaired insulin action within skeletal muscle, adipose tissue, and the liver is an important characteristic of type 2 diabetes (T2D). In order to identify common underlying defects in insulin-sensitive tissues that may be involved in the pathogenesis of T2D, the gene expression profiles of skeletal muscle, visceral adipose tissue, and liver from autopsy donors with or without T2D were examined using oligonucleotide microarrays and quantitative reverse transcriptase-PCR. Compared with controls, 691 genes were commonly dysregulated in these three insulin-sensitive tissues of humans with T2D. These co-expressed genes were enriched within the mitochondrion, with suggested involvement in energy metabolic processes such as glycolysis and gluconeogenesis, fatty acid beta oxidative, tricarboxylic acid cycle, and electron transport. Genes related to energy metabolism were mostly downregulated in diabetic skeletal muscle and visceral adipose tissue, while they were upregulated in the diabetic liver. This observed dysregulation in energy-related metabolism may be the underlying factor leading to the molecular mechanisms responsible for the insulin resistance of patients with T2D.

A curated collection of tissue microarray images and clinical outcome data of prostate cancer patients

PubMed Central

Zhong, Qing; Guo, Tiannan; Rechsteiner, Markus; Rüschoff, Jan H.; Rupp, Niels; Fankhauser, Christian; Saba, Karim; Mortezavi, Ashkan; Poyet, Cédric; Hermanns, Thomas; Zhu, Yi; Moch, Holger; Aebersold, Ruedi; Wild, Peter J.

2017-01-01

Microscopy image data of human cancers provide detailed phenotypes of spatially and morphologically intact tissues at single-cell resolution, thus complementing large-scale molecular analyses, e.g., next generation sequencing or proteomic profiling. Here we describe a high-resolution tissue microarray (TMA) image dataset from a cohort of 71 prostate tissue samples, which was hybridized with bright-field dual colour chromogenic and silver in situ hybridization probes for the tumour suppressor gene PTEN. These tissue samples were digitized and supplemented with expert annotations, clinical information, statistical models of PTEN genetic status, and computer source codes. For validation, we constructed an additional TMA dataset for 424 prostate tissues, hybridized with FISH probes for PTEN, and performed survival analysis on a subset of 339 radical prostatectomy specimens with overall, disease-specific and recurrence-free survival (maximum 167 months). For application, we further produced 6,036 image patches derived from two whole slides. Our curated collection of prostate cancer data sets provides reuse potential for both biomedical and computational studies. PMID:28291248

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

NASA Technical Reports Server (NTRS)

Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

2001-01-01

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species.

PubMed

Vallée, Maud; Gravel, Catherine; Palin, Marie-France; Reghenas, Hélène; Stothard, Paul; Wishart, David S; Sirard, Marc-André

2005-07-01

The main objective of the present study was to identify novel oocyte-specific genes in three different species: bovine, mouse, and Xenopus laevis. To achieve this goal, two powerful technologies were combined: a polymerase chain reaction (PCR)-based cDNA subtraction, and cDNA microarrays. Three subtractive libraries consisting of 3456 clones were established and enriched for oocyte-specific transcripts. Sequencing analysis of the positive insert-containing clones resulted in the following classification: 53% of the clones corresponded to known cDNAs, 26% were classified as uncharacterized cDNAs, and a final 9% were classified as novel sequences. All these clones were used for cDNA microarray preparation. Results from these microarray analyses revealed that in addition to already known oocyte-specific genes, such as GDF9, BMP15, and ZP, known genes with unknown function in the oocyte were identified, such as a MLF1-interacting protein (MLF1IP), B-cell translocation gene 4 (BTG4), and phosphotyrosine-binding protein (xPTB). Furthermore, 15 novel oocyte-specific genes were validated by reverse transcription-PCR to confirm their preferential expression in the oocyte compared to somatic tissues. The results obtained in the present study confirmed that microarray analysis is a robust technique to identify true positives from the suppressive subtractive hybridization experiment. Furthermore, obtaining oocyte-specific genes from three species simultaneously allowed us to look at important genes that are conserved across species. Further characterization of these novel oocyte-specific genes will lead to a better understanding of the molecular mechanisms related to the unique functions found in the oocyte.

A New Way to Introduce Microarray Technology in a Lecture/Laboratory Setting by Studying the Evolution of This Modern Technology

ERIC Educational Resources Information Center

Rowland-Goldsmith, Melissa

2009-01-01

DNA microarray is an ordered grid containing known sequences of DNA, which represent many of the genes in a particular organism. Each DNA sequence is unique to a specific gene. This technology enables the researcher to screen many genes from cells or tissue grown in different conditions. We developed an undergraduate lecture and laboratory…

ExprAlign - the identification of ESTs in non-model species by alignment of cDNA microarray expression profiles

PubMed Central

2009-01-01

Background Sequence identification of ESTs from non-model species offers distinct challenges particularly when these species have duplicated genomes and when they are phylogenetically distant from sequenced model organisms. For the common carp, an environmental model of aquacultural interest, large numbers of ESTs remained unidentified using BLAST sequence alignment. We have used the expression profiles from large-scale microarray experiments to suggest gene identities. Results Expression profiles from ~700 cDNA microarrays describing responses of 7 major tissues to multiple environmental stressors were used to define a co-expression landscape. This was based on the Pearsons correlation coefficient relating each gene with all other genes, from which a network description provided clusters of highly correlated genes as 'mountains'. We show that these contain genes with known identities and genes with unknown identities, and that the correlation constitutes evidence of identity in the latter. This procedure has suggested identities to 522 of 2701 unknown carp ESTs sequences. We also discriminate several common carp genes and gene isoforms that were not discriminated by BLAST sequence alignment alone. Precision in identification was substantially improved by use of data from multiple tissues and treatments. Conclusion The detailed analysis of co-expression landscapes is a sensitive technique for suggesting an identity for the large number of BLAST unidentified cDNAs generated in EST projects. It is capable of detecting even subtle changes in expression profiles, and thereby of distinguishing genes with a common BLAST identity into different identities. It benefits from the use of multiple treatments or contrasts, and from the large-scale microarray data. PMID:19939286

Intratumoral immune cells expressing PD-1/PD-L1 and their prognostic implications in cancer: a meta-analysis.

PubMed

Kim, Younghoon; Wen, Xianyu; Cho, Nam Yun; Kang, Gyeong Hoon

2018-05-01

The prognostic value of immune cells expressing programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) in cancer are controversial, and the potential differential impact of using tissue microarrays and whole tissue sections to assess the positivity of immune cells has not been addressed. The current study included 30 eligible studies with 7251 patients that evaluated the relationship between tumor-infiltrating lymphocytes expressing PD-1/PD-L1 and overall survival and disease-free survival, or progression-free survival. Subgroup analysis was based on the tissue type of cancer and the type of tissue sampling (tissue microarray or whole tissue section). In the meta-analysis, PD-1-positive and PD-L1-positive tumor-infiltrating lymphocytes had a positive effect on disease-free survival or progression-free survival (hazard ratio [HR] 0.732; 95% confidence interval [CI] 0.565, 0.947; and HR 0.727; 95% CI 0.584, 0.905, respectively). PD-L1-positive tumor-infiltrating lymphocytes had a positive impact on overall survival in studies using tissue microarray (HR 0.586; 95% CI 0.476, 0.721), but had a poor impact when only whole tissue sections were considered (HR 1.558; 95% CI 1.232, 1.969). Lung cancer was associated with good overall survival and disease-free survival (HR 0.639; 95% CI 0.491, 0.831; and HR 0.693; 95% CI 0.538, 0.891, respectively) for PD-1-positive tumor-infiltrating lymphocytes, and colorectal cancer showed favorable disease-free survival (HR 0.471; 95% CI 0.308, 0.722) for PD-L1-positive tumor-infiltrating lymphocytes. Immune cells expressing PD-1 and PD-L1 within tumors are associated with the prognosis. However, the correlation may vary among different tumor types and by the type of tissue sampling used for the assessment.

Differential prioritization between relevance and redundancy in correlation-based feature selection techniques for multiclass gene expression data.

PubMed

Ooi, Chia Huey; Chetty, Madhu; Teng, Shyh Wei

2006-06-23

Due to the large number of genes in a typical microarray dataset, feature selection looks set to play an important role in reducing noise and computational cost in gene expression-based tissue classification while improving accuracy at the same time. Surprisingly, this does not appear to be the case for all multiclass microarray datasets. The reason is that many feature selection techniques applied on microarray datasets are either rank-based and hence do not take into account correlations between genes, or are wrapper-based, which require high computational cost, and often yield difficult-to-reproduce results. In studies where correlations between genes are considered, attempts to establish the merit of the proposed techniques are hampered by evaluation procedures which are less than meticulous, resulting in overly optimistic estimates of accuracy. We present two realistically evaluated correlation-based feature selection techniques which incorporate, in addition to the two existing criteria involved in forming a predictor set (relevance and redundancy), a third criterion called the degree of differential prioritization (DDP). DDP functions as a parameter to strike the balance between relevance and redundancy, providing our techniques with the novel ability to differentially prioritize the optimization of relevance against redundancy (and vice versa). This ability proves useful in producing optimal classification accuracy while using reasonably small predictor set sizes for nine well-known multiclass microarray datasets. For multiclass microarray datasets, especially the GCM and NCI60 datasets, DDP enables our filter-based techniques to produce accuracies better than those reported in previous studies which employed similarly realistic evaluation procedures.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Cancer testis antigen OY-TES-1: analysis of protein expression in ovarian cancer with tissue microarrays.

PubMed

Fan, R; Huang, W; Luo, B; Zhang, Q M; Xiao, S W; Xie, X X

2015-01-01

Revised manuscript accepted for publication March 5, Objectives: The purpose of this study was to determine the potential of cancer testis antigen OY-TES-1 as a vaccine for ovarian cancer (OC). A tissue microarray (TMA) containing 107 samples from OC tissues and 48 samples from OC adjacent tissues was analyzed by immunohistochemistry with the OY-TES-1 polyclonal antibody. The correlation between OY-TES-1 and clinic pathological traits of OC was statistically analyzed. The expression of OY-TES-1 protein was found in 81% (87/107) of OC tissues and 56% (27/48) of OC adjacent tissues. The immunostaining intensity of OY-TES-1 in OC tissues was significantly higher than that in OC adjacent tissues tested (p = 0.040). OC adjacent tissues only demonstrated lower immunostaining intensity, whereas some of OC tissues presented higher immunostaining intensity and majority showed the heterogeneity of protein distribution. There was no statistically significant correlation found between OY-TES-1 expression and any other clinicopathological traits such as age, FIGO stage, pathological grade, and histological type. OY-TES-1 was expressed in OC tissues with a high proportion, and some of OC tissues presented OY-TES-1 expression in high level vs OC adjacent tissues. OY-TES-1 could be an attractive target for immunotherapy for OC in the future.

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Standardized Scalp Massage Results in Increased Hair Thickness by Inducing Stretching Forces to Dermal Papilla Cells in the Subcutaneous Tissue

PubMed Central

Kobayashi, Kazuhiro; Hama, Takanori; Murakami, Kasumi; Ogawa, Rei

2016-01-01

Objective: In this study, we evaluated the effect of scalp massage on hair in Japanese males and the effect of stretching forces on human dermal papilla cells in vitro. Methods: Nine healthy men received 4 minutes of standardized scalp massage per day for 24 weeks using a scalp massage device. Total hair number, hair thickness, and hair growth rate were evaluated. The mechanical effect of scalp massage on subcutaneous tissue was analyzed using a finite element method. To evaluate the effect of mechanical forces, human dermal papilla cells were cultured using a 72-hour stretching cycle. Gene expression change was analyzed using DNA microarray analyses. In addition, expression of hair cycle-related genes including IL6, NOGGIN, BMP4, and SMAD4 were evaluated using real-time reverse transcription-polymerase chain reaction. Results: Standardized scalp massage resulted in increased hair thickness 24 weeks after initiation of massage (0.085 ± 0.003 mm vs 0.092 ± 0.001 mm). Finite element method showed that scalp massage caused z-direction displacement and von Mises stress on subcutaneous tissue. In vitro, DNA microarray showed gene expression change significantly compared with nonstretching human dermal papilla cells. A total of 2655 genes were upregulated and 2823 genes were downregulated. Real-time reverse transcription-polymerase chain reaction demonstrated increased expression of hair cycle–related genes such as NOGGIN, BMP4, SMAD4, and IL6ST and decrease in hair loss–related genes such as IL6. Conclusions: Stretching forces result in changes in gene expression in human dermal papilla cells. Standardized scalp massage is a way to transmit mechanical stress to human dermal papilla cells in subcutaneous tissue. Hair thickness was shown to increase with standardized scalp massage. PMID:26904154

Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer.

PubMed

Beyer, Sasha J; Zhang, Xiaoli; Jimenez, Rafael E; Lee, Mei-Ling T; Richardson, Andrea L; Huang, Kun; Jhiang, Sissy M

2011-10-11

Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses

PubMed Central

Sforzini, Susanna; Arlt, Volker M.; Barranger, Audrey; Dallas, Lorna J.; Oliveri, Caterina; Aminot, Yann; Pacchioni, Beniamina; Millino, Caterina; Lanfranchi, Gerolamo; Readman, James W.; Moore, Michael N.; Viarengo, Aldo; Jha, Awadhesh N.

2017-01-01

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new ‘STressREsponse Microarray’ (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 μg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota. PMID:28651000

Recommendations for the use of microarrays in prenatal diagnosis.

PubMed

Suela, Javier; López-Expósito, Isabel; Querejeta, María Eugenia; Martorell, Rosa; Cuatrecasas, Esther; Armengol, Lluis; Antolín, Eugenia; Domínguez Garrido, Elena; Trujillo-Tiebas, María José; Rosell, Jordi; García Planells, Javier; Cigudosa, Juan Cruz

2017-04-07

Microarray technology, recently implemented in international prenatal diagnosis systems, has become one of the main techniques in this field in terms of detection rate and objectivity of the results. This guideline attempts to provide background information on this technology, including technical and diagnostic aspects to be considered. Specifically, this guideline defines: the different prenatal sample types to be used, as well as their characteristics (chorionic villi samples, amniotic fluid, fetal cord blood or miscarriage tissue material); variant reporting policies (including variants of uncertain significance) to be considered in informed consents and prenatal microarray reports; microarray limitations inherent to the technique and which must be taken into account when recommending microarray testing for diagnosis; a detailed clinical algorithm recommending the use of microarray testing and its introduction into routine clinical practice within the context of other genetic tests, including pregnancies in families with a genetic history or specific syndrome suspicion, first trimester increased nuchal translucency or second trimester heart malformation and ultrasound findings not related to a known or specific syndrome. This guideline has been coordinated by the Spanish Association for Prenatal Diagnosis (AEDP, «Asociación Española de Diagnóstico Prenatal»), the Spanish Human Genetics Association (AEGH, «Asociación Española de Genética Humana») and the Spanish Society of Clinical Genetics and Dysmorphology (SEGCyD, «Sociedad Española de Genética Clínica y Dismorfología»). Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis

PubMed Central

Loftus, S. K.; Chen, Y.; Gooden, G.; Ryan, J. F.; Birznieks, G.; Hilliard, M.; Baxevanis, A. D.; Bittner, M.; Meltzer, P.; Trent, J.; Pavan, W.

1999-01-01

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 × 10−9). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 × 10−8). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases. PMID:10430933

Immune and inflammatory gene signature in rat cerebrum in subarachnoid hemorrhage with microarray analysis.

PubMed

Lee, Chu-I; Chou, An-Kuo; Lin, Ching-Chih; Chou, Chia-Hua; Loh, Joon-Khim; Lieu, Ann-Shung; Wang, Chih-Jen; Huang, Chi-Ying F; Howng, Shen-Long; Hong, Yi-Ren

2012-01-01

Cerebral vasospasm following subarachnoid hemorrhage (SAH) has been studied in terms of a contraction of the major cerebral arteries, but the effect of cerebrum tissue in SAH is not yet well understood. To gain insight into the biology of SAH-expressing cerebrum, we employed oligonucleotide microarrays to characterize the gene expression profiles of cerebrum tissue at the early stage of SAH. Functional gene expression in the cerebrum was analyzed 2 h following stage 1-hemorrhage in Sprague-Dawley rats. mRNA was investigated by performing microarray and quantitative real-time PCR analyses, and protein expression was determined by Western blot analysis. In this study, 18 upregulated and 18 downregulated genes displayed at least a 1.5-fold change. Five genes were verified by real-time PCR, including three upregulated genes [prostaglandin E synthase (PGES), CD14 antigen, and tissue inhibitor of metalloproteinase 1 (TIMP1)] as well as two downregulated genes [KRAB-zinc finger protein-2 (KZF-2) and γ-aminobutyric acid B receptor 1 (GABA B receptor)]. Notably, there were functional implications for the three upregulated genes involved in the inflammatory SAH process. However, the mechanisms leading to decreased KZF-2 and GABA B receptor expression in SAH have never been characterized. We conclude that oligonucleotide microarrays have the potential for use as a method to identify candidate genes associated with SAH and to provide novel investigational targets, including genes involved in the immune and inflammatory response. Furthermore, understanding the regulation of MMP9/TIMP1 during the early stages of SAH may elucidate the pathophysiological mechanisms in SAH rats.

An 80-year experience with optic nerve glioma cases at the Armed Forces Institute of Pathology: evolution from museum to molecular evaluation suggests possibe interventions in the cellular senescence and microglial pathways (an American Ophthalmological Society thesis).

PubMed

Cameron, J Douglas; Rodriguez, Fausto J; Rushing, Elisabeth; Horkayne-Szakaly, Iren; Eberhart, Charles

2014-01-01

To determine whether p16, a molecular marker of cellular senescence, and CD68, a microglial marker, are detectible in optic nerve glioma tissue stored for decades, thus providing potential targets for pharmacologic intervention. Cases were retrieved from the Armed Forces Institute of Pathology Registry of Ophthalmic Pathology. Clinical information was tabulated. In specimens with sufficient tissue, a tissue microarray was constructed to conduct molecular studies. Ninety-two cases were included: gender distribution was in a ratio of one male to 1.6 females, and age range was 2 months to 50 years (average age, 10.8 years). Neurofibromatosis type 1 was identified in 10 cases (10.8%). The majority presented with decreased vision and exophthalmos. Forty-eight cases were studied by a tissue microarray construction. Glial fibrillary acidic protein, a control for immunoreactivity, was positive in 46 cases (96%). Immunoreactivity for p16 protein was seen in 36 cases (75%) and CD68-positive cells in 34 (71%). Limitations include referral bias, limited clinical information, limited amount of tissue, and extended period of tissue preservation. Optic nerve glioma is a tumor of the visual axis in young individuals, which is generally indolent but with a variable clinical course. Traditional histopathologic techniques have not been reliably predictive of clinical course. This microarray contains tumors with representative demographic, clinical, and histologic characteristics for optic nerve glioma. Immunoreactivity for p16 protein and CD68 is positive in the majority. These findings suggest a possible explanation for the variable clinical course and identify therapeutic targets in the cell senescence and microglial pathways.

Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression

PubMed Central

2011-01-01

Introduction Pokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms. Methods Tissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene. Results Pokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P < 0.0001). ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter. Conclusions Pokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy. PMID:21392388

RankProd Combined with Genetic Algorithm Optimized Artificial Neural Network Establishes a Diagnostic and Prognostic Prediction Model that Revealed C1QTNF3 as a Biomarker for Prostate Cancer.

PubMed

Hou, Qi; Bing, Zhi-Tong; Hu, Cheng; Li, Mao-Yin; Yang, Ke-Hu; Mo, Zu; Xie, Xiang-Wei; Liao, Ji-Lin; Lu, Yan; Horie, Shigeo; Lou, Ming-Wu

2018-06-01

Prostate cancer (PCa) is the most commonly diagnosed cancer in males in the Western world. Although prostate-specific antigen (PSA) has been widely used as a biomarker for PCa diagnosis, its results can be controversial. Therefore, new biomarkers are needed to enhance the clinical management of PCa. From publicly available microarray data, differentially expressed genes (DEGs) were identified by meta-analysis with RankProd. Genetic algorithm optimized artificial neural network (GA-ANN) was introduced to establish a diagnostic prediction model and to filter candidate genes. The diagnostic and prognostic capability of the prediction model and candidate genes were investigated in both GEO and TCGA datasets. Candidate genes were further validated by qPCR, Western Blot and Tissue microarray. By RankProd meta-analyses, 2306 significantly up- and 1311 down-regulated probes were found in 133 cases and 30 controls microarray data. The overall accuracy rate of the PCa diagnostic prediction model, consisting of a 15-gene signature, reached up to 100% in both the training and test dataset. The prediction model also showed good results for the diagnosis (AUC = 0.953) and prognosis (AUC of 5 years overall survival time = 0.808) of PCa in the TCGA database. The expression levels of three genes, FABP5, C1QTNF3 and LPHN3, were validated by qPCR. C1QTNF3 high expression was further validated in PCa tissue by Western Blot and Tissue microarray. In the GEO datasets, C1QTNF3 was a good predictor for the diagnosis of PCa (GSE6956: AUC = 0.791; GSE8218: AUC = 0.868; GSE26910: AUC = 0.972). In the TCGA database, C1QTNF3 was significantly associated with PCa patient recurrence free survival (P < .001, AUC = 0.57). In this study, we have developed a diagnostic and prognostic prediction model for PCa. C1QTNF3 was revealed as a promising biomarker for PCa. This approach can be applied to other high-throughput data from different platforms for the discovery of oncogenes or biomarkers in different kinds of diseases. Copyright © 2018. Published by Elsevier B.V.

Overcoming confounded controls in the analysis of gene expression data from microarray experiments.

PubMed

Bhattacharya, Soumyaroop; Long, Dang; Lyons-Weiler, James

2003-01-01

A potential limitation of data from microarray experiments exists when improper control samples are used. In cancer research, comparisons of tumour expression profiles to those from normal samples is challenging due to tissue heterogeneity (mixed cell populations). A specific example exists in a published colon cancer dataset, in which tissue heterogeneity was reported among the normal samples. In this paper, we show how to overcome or avoid the problem of using normal samples that do not derive from the same tissue of origin as the tumour. We advocate an exploratory unsupervised bootstrap analysis that can reveal unexpected and undesired, but strongly supported, clusters of samples that reflect tissue differences instead of tumour versus normal differences. All of the algorithms used in the analysis, including the maximum difference subset algorithm, unsupervised bootstrap analysis, pooled variance t-test for finding differentially expressed genes and the jackknife to reduce false positives, are incorporated into our online Gene Expression Data Analyzer ( http:// bioinformatics.upmc.edu/GE2/GEDA.html ).

Intratumoral concentration of estrogens and clinicopathological changes in ductal carcinoma in situ following aromatase inhibitor letrozole treatment

PubMed Central

Takagi, K; Ishida, T; Miki, Y; Hirakawa, H; Kakugawa, Y; Amano, G; Ebata, A; Mori, N; Nakamura, Y; Watanabe, M; Amari, M; Ohuchi, N; Sasano, H; Suzuki, T

2013-01-01

Background: Estrogens have important roles in ductal carcinoma in situ (DCIS) of the breast. However, the significance of presurgical aromatase inhibitor treatment remains unclear. Therefore, we examined intratumoral concentration of estrogens and changes of clinicopathological factors in DCIS after letrozole treatment. Methods: Ten cases of postmenopausal oestrogen receptor (ER)-positive DCIS were examined. They received oral letrozole before the surgery, and the tumour size was evaluated by ultrasonography. Surgical specimens and corresponding biopsy samples were used for immunohistochemistry. Snap-frozen specimens were also available in a subset of cases, and used for hormone assays and microarray analysis. Results: Intratumoral oestrogen levels were significantly lower in DCIS treated with letrozole compared with that in those without the therapy. A great majority of oestrogen-induced genes showed low expression levels in DCIS treated with letrozole by microarray analysis. Moreover, letrozole treatment reduced the greatest dimension of DCIS, and significantly decreased Ki-67 and progesterone receptor immunoreactivity in DCIS tissues. Conclusion: These results suggest that estrogens are mainly produced by aromatase in DCIS tissues, and aromatase inhibitors potently inhibit oestrogen actions in postmenopausal ER-positive DCIS through rapid deprivation of intratumoral estrogens. PMID:23756858

Gene profiling, biomarkers and pathways characterizing HCV-related hepatocellular carcinoma

PubMed Central

De Giorgi, Valeria; Monaco, Alessandro; Worchech, Andrea; Tornesello, MariaLina; Izzo, Francesco; Buonaguro, Luigi; Marincola, Francesco M; Wang, Ena; Buonaguro, Franco M

2009-01-01

Background Hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The molecular mechanisms of HCV-induced hepatocarcinogenesis are not yet fully elucidated. Besides indirect effects as tissue inflammation and regeneration, a more direct oncogenic activity of HCV can be postulated leading to an altered expression of cellular genes by early HCV viral proteins. In the present study, a comparison of gene expression patterns has been performed by microarray analysis on liver biopsies from HCV-positive HCC patients and HCV-negative controls. Methods Gene expression profiling of liver tissues has been performed using a high-density microarray containing 36'000 oligos, representing 90% of the human genes. Samples were obtained from 14 patients affected by HCV-related HCC and 7 HCV-negative non-liver-cancer patients, enrolled at INT in Naples. Transcriptional profiles identified in liver biopsies from HCC nodules and paired non-adjacent non-HCC liver tissue of the same HCV-positive patients were compared to those from HCV-negative controls by the Cluster program. The pathway analysis was performed using the BRB-Array- Tools based on the "Ingenuity System Database". Significance threshold of t-test was set at 0.001. Results Significant differences were found between the expression patterns of several genes falling into different metabolic and inflammation/immunity pathways in HCV-related HCC tissues as well as the non-HCC counterpart compared to normal liver tissues. Only few genes were found differentially expressed between HCV-related HCC tissues and paired non-HCC counterpart. Conclusion In this study, informative data on the global gene expression pattern of HCV-related HCC and non-HCC counterpart, as well as on their difference with the one observed in normal liver tissues have been obtained. These results may lead to the identification of specific biomarkers relevant to develop tools for detection, diagnosis, and classification of HCV-related HCC. PMID:19821982

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

PubMed Central

Robertson, David; Savage, Kay; Reis-Filho, Jorge S; Isacke, Clare M

2008-01-01

Background Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples. PMID:18366689

Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation.

PubMed

Richard, Arianne C; Lyons, Paul A; Peters, James E; Biasci, Daniele; Flint, Shaun M; Lee, James C; McKinney, Eoin F; Siegel, Richard M; Smith, Kenneth G C

2014-08-04

Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study. Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this "gold-standard" comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues. Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.

The tissue microarray data exchange specification: A document type definition to validate and enhance XML data

PubMed Central

Nohle, David G; Ayers, Leona W

2005-01-01

Background The Association for Pathology Informatics (API) Extensible Mark-up Language (XML) TMA Data Exchange Specification (TMA DES) proposed in April 2003 provides a community-based, open source tool for sharing tissue microarray (TMA) data in a common format. Each tissue core within an array has separate data including digital images; therefore an organized, common approach to produce, navigate and publish such data facilitates viewing, sharing and merging TMA data from different laboratories. The AIDS and Cancer Specimen Resource (ACSR) is a HIV/AIDS tissue bank consortium sponsored by the National Cancer Institute (NCI) Division of Cancer Treatment and Diagnosis (DCTD). The ACSR offers HIV-related malignancies and uninfected control tissues in microarrays (TMA) accompanied by de-identified clinical data to approved researchers. Exporting our TMA data into the proposed API specified format offers an opportunity to evaluate the API specification in an applied setting and to explore its usefulness. Results A document type definition (DTD) that governs the allowed common data elements (CDE) in TMA DES export XML files was written, tested and evolved and is in routine use by the ACSR. This DTD defines TMA DES CDEs which are implemented in an external file that can be supplemented by internal DTD extensions for locally defined TMA data elements (LDE). Conclusion ACSR implementation of the TMA DES demonstrated the utility of the specification and allowed application of a DTD to validate the language of the API specified XML elements and to identify possible enhancements within our TMA data management application. Improvements to the specification have additionally been suggested by our experience in importing other institution's exported TMA data. Enhancements to TMA DES to remove ambiguous situations and clarify the data should be considered. Better specified identifiers and hierarchical relationships will make automatic use of the data possible. Our tool can be used to reorder data and add identifiers; upgrading data for changes in the specification can be automatically accomplished. Using a DTD (optionally reflecting our proposed enhancements) can provide stronger validation of exported TMA data. PMID:15871741

No-cost manual method for preparation of tissue microarrays having high quality comparable to semiautomated methods.

PubMed

Foda, Abd Al-Rahman Mohammad

2013-05-01

Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl's technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories.

The prognostic implication of the expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in primary locally advanced oral squamous cell carcinoma cases: a tissue microarray study.

PubMed

Solomon, Monica Charlotte; Vidyasagar, M S; Fernandes, Donald; Guddattu, Vasudev; Mathew, Mary; Shergill, Ankur Kaur; Carnelio, Sunitha; Chandrashekar, Chetana

2016-12-01

Oral squamous cell carcinomas comprise a heterogeneous tumor cell population with varied molecular characteristics, which makes prognostication of these tumors a complex and challenging issue. Thus, molecular profiling of these tumors is advantageous for an accurate prognostication and treatment planning. This is a retrospective study on a cohort of primary locally advanced oral squamous cell carcinomas (n = 178) of an Indian rural population. The expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of primary locally advanced oral squamous cell carcinomas was evaluated. A potential biomarker that can predict the tumor response to treatment was identified. Formalin-fixed paraffin-embedded tumor blocks of (n = 178) of histopathologically diagnosed cases of locally advanced oral squamous cell carcinomas were selected. Tissue microarray blocks were constructed with 2 cores of 2 mm diameter from each tumor block. Four-micron-thick sections were cut from these tissue microarray blocks. These tissue microarray sections were immunohistochemically stained for EGFR, p53, Bcl-2, cyclin D1 and p16. In this cohort, EGFR was the most frequently expressed 150/178 (84%) biomarker of the cases. Kaplan-Meier analysis showed a significant association (p = 0.038) between expression of p53 and a poor prognosis. A Poisson regression analysis showed that tumors that expressed p53 had a two times greater chance of recurrence (unadjusted IRR-95% CI 2.08 (1.03, 4.5), adjusted IRR-2.29 (1.08, 4.8) compared with the tumors that did not express this biomarker. Molecular profiling of oral squamous cell carcinomas will enable us to categorize our patients into more realistic risk groups. With biologically guided tumor characterization, personalized treatment protocols can be designed for individual patients, which will improve the quality of life of these patients.

Long noncoding RNA OR3A4 promotes metastasis and tumorigenicity in gastric cancer

PubMed Central

Guo, Xiaobo; Yang, Ziguo; Zhi, Qiaoming; Wang, Dan; Guo, Lei; Li, Guimei; Miao, Ruizhen; Shi, Yulong; Kuang, Yuting

2016-01-01

The contribution of long noncoding RNAs (lncRNAs) to metastasis of gastric cancer remains largely unknown. We used microarray analysis to identify lncRNAs differentially expressed between normal gastric tissues and gastric cancer tissues and validated these differences in quantitative real-time (qRT)-PCR experiments. The expression levels of lncRNA olfactory receptor, family 3, subfamily A, member 4 (OR3A4) were significantly associated with lymphatic metastasis, the depth of cancer invasion, and distal metastasis in 130 paired gastric cancer tissues. The effects of OR3A4 were assessed by overexpressing and silencing OR3A4 in gastric cancer cells. OR3A4 promoted cancer cell growth, angiogenesis, metastasis, and tumorigenesis in vitro and in vivo. Global microarray analysis combined with RT-PCR, RNA immunoprecipitation, and RNA pull-down analyses after OR3A4 transfection demonstrated that OR3A4 influenced biologic functions in gastric cancer cells via regulating the activation of PDLIM2, MACC1, NTN4, and GNB2L1. Our results reveal OR3A4 as an oncogenic lncRNA that promotes tumor progression, Therefore, lncRNAs might function as key regulatory hubs in gastric cancer progression. PMID:26863570

A tissue microarray study of toll-like receptor 4, decoy receptor 3, and external signal regulated kinase 1/2 expressions in astrocytoma.

PubMed

Lin, Chih-Kung; Ting, Chun-Chieh; Tsai, Wen-Chiuan; Chen, Yuan-Wu; Hueng, Dueng-Yuan

2016-01-01

Decoy receptor 3 (DcR3) functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor family. It is highly expressed in many tumors and its expression can be regulated by the MAPK/ERK signaling pathway and ERK is a vital member of this pathway. Toll-like receptor 4 (TLR4) is expressed on immune cells. Increased TLR4 expression has been associated with various types of cancers. The study was conducted to investigate the expression of DcR3, ERK1/2, and TLR4 in astrocytomas and evaluate if they are validating markers for discriminating glioblastoma from anaplastic astrocytoma in limited surgical specimen. Expression of DcR3, ERK1/2, and TLR4 was determined by immunohistochemical staining of tissue microarray from 48 paraffin-embedded tissues. A binary logistic regression method was used to generate functions that discriminate between anaplastic astrocytomas and glioblastomas. The expression of TLR4 and DcR3 was significantly higher in glioblastomas than in anaplastic astrocytomas. DcR3 could discriminate anaplastic astrocytomas from glioblastomas with high sensitivity (93.8%), specificity (90%), and accuracy (92.3%). Our results suggest that DcR3 may be a useful marker for discriminating anaplastic astrocytomas from glioblastomas.

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

PubMed Central

Pinzani, Pamela; Mancini, Irene; Vinci, Serena; Chiari, Marcella; Orlando, Claudio; Cremonesi, Laura; Ferrari, Maurizio

2013-01-01

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy. PMID:23536897

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

PubMed

Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

2004-12-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

Genetic and Epigenetic Biomarkers for Recurrent Prostate Cancer After Radiotherapy

DTIC Science & Technology

2015-07-01

several distinct advantages over surgical treatment, such as no complications from surgery, and a low risk of urinary incontinence , RT treatment takes...Khorana, Tissue factor and VEGF expression in prostate carcinoma: a tissue microarray study. Cancer Invest, 2009. 27(4): p. 430-4. 7. Crawford, E.D

Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray

PubMed Central

Kennedy, Laura; Vass, J. Keith; Haggart, D. Ross; Moore, Steve; Burczynski, Michael E.; Crowther, Dan; Miele, Gino

2008-01-01

Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene™ RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2™ enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene™ blood samples also advocate a short, fixed room temperature storage time for all PAXgene™ blood samples collected for the purposes of global transcriptional profiling in clinical studies. PMID:19578521

The 'PUCE CAFE' Project: the First 15K Coffee Microarray, a New Tool for Discovering Candidate Genes correlated to Agronomic and Quality Traits

PubMed Central

2011-01-01

Background Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research. PMID:21208403

Hybrid feature selection algorithm using symmetrical uncertainty and a harmony search algorithm

NASA Astrophysics Data System (ADS)

Salameh Shreem, Salam; Abdullah, Salwani; Nazri, Mohd Zakree Ahmad

2016-04-01

Microarray technology can be used as an efficient diagnostic system to recognise diseases such as tumours or to discriminate between different types of cancers in normal tissues. This technology has received increasing attention from the bioinformatics community because of its potential in designing powerful decision-making tools for cancer diagnosis. However, the presence of thousands or tens of thousands of genes affects the predictive accuracy of this technology from the perspective of classification. Thus, a key issue in microarray data is identifying or selecting the smallest possible set of genes from the input data that can achieve good predictive accuracy for classification. In this work, we propose a two-stage selection algorithm for gene selection problems in microarray data-sets called the symmetrical uncertainty filter and harmony search algorithm wrapper (SU-HSA). Experimental results show that the SU-HSA is better than HSA in isolation for all data-sets in terms of the accuracy and achieves a lower number of genes on 6 out of 10 instances. Furthermore, the comparison with state-of-the-art methods shows that our proposed approach is able to obtain 5 (out of 10) new best results in terms of the number of selected genes and competitive results in terms of the classification accuracy.

Genomic expression patterns of cardiac tissues from dogs with dilated cardiomyopathy.

PubMed

Oyama, Mark A; Chittur, Sridar

2005-07-01

To evaluate global genome expression patterns of left ventricular tissues from dogs with dilated cardiomyopathy (DCM). Tissues obtained from the left ventricle of 2 Doberman Pinschers with end-stage DCM and 5 healthy control dogs. Transcriptional activities of 23,851 canine DNA sequences were determined by use of an oligonucleotide microarray. Genome expression patterns of DCM tissue were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with gene expression for tissues from 5 healthy control dogs. 478 transcripts were differentially expressed (> or = 2.5-fold change). In DCM tissue, expression of 173 transcripts was upregulated and expression of 305 transcripts was downregulated, compared with expression for control tissues. Of the 478 transcripts, 167 genes could be specifically identified. These genes were grouped into 1 of 8 categories on the basis of their primary physiologic function. Grouping revealed that pathways involving cellular energy production, signaling and communication, and cell structure were generally downregulated, whereas pathways involving cellular defense and stress responses were upregulated. Many previously unreported genes that may contribute to the pathophysiologic aspects of heart disease were identified. Evaluation of global expression patterns provides a molecular portrait of heart failure, yields insights into the pathophysiologic aspects of DCM, and identifies intriguing genes and pathways for further study.

Biomarkers of Selenium Action in Prostate Cancer

DTIC Science & Technology

2005-01-01

secretory by conventional methods according to published literature. In addition, we have determined the similarities and differences in global gene...transition zone tissue of a 42-year-old man ac- arrays in the resulting data tables were ordered by their cording to previously described methods [4]. The pre...hundred fifteen genes identified by ELISA method . Replicating the conditions used for the SAM analysis showed significant differential expres- microarray

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

PubMed Central

2011-01-01

Background It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Methods Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. Results The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Conclusions Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A-bomb radiation may affect the increased amount of CNA as a hallmark of GIN and, subsequently, be associated with a higher histologic grade in breast cancer found in A-bomb survivors. PMID:22152285

Circular RNA and gene expression profiles in gastric cancer based on microarray chip technology.

PubMed

Sui, Weiguo; Shi, Zhoufang; Xue, Wen; Ou, Minglin; Zhu, Ying; Chen, Jiejing; Lin, Hua; Liu, Fuhua; Dai, Yong

2017-03-01

The aim of the present study was to screen gastric cancer (GC) tissue and adjacent tissue for differences in mRNA and circular (circRNA) expression, to analyze the differences in circRNA and mRNA expression, and to investigate the circRNA expression in gastric carcinoma and its mechanism. circRNA and mRNA differential expression profiles generated using Agilent microarray technology were analyzed in the GC tissues and adjacent tissues. qRT-PCR was used to verify the differential expression of circRNAs and mRNAs according to the interactions between circRNAs and miRNAs as well as the possible existence of miRNA and mRNA interactions. We found that: i) the circRNA expression profile revealed 1,285 significant differences in circRNA expression, with circRNA expression downregulated in 594 samples and upregulated in 691 samples via interactions with miRNAs. The qRT-PCR validation experiments showed that hsa_circRNA_400071, hsa_circRNA_000543 and hsa_circRNA_001959 expression was consistent with the microarray analysis results. ii) 29,112 genes were found in the GC tissues and adjacent tissues, including 5,460 differentially expressed genes. Among them, 2,390 differentially expressed genes were upregulated and 3,070 genes were downregulated. Gene Ontology (GO) analysis of the differentially expressed genes revealed these genes involved in biological process classification, cellular component classification and molecular function classification. Pathway analysis of the differentially expressed genes identified 83 significantly enriched genes, including 28 upregulated genes and 55 downregulated genes. iii) 69 differentially expressed circRNAs were found that might adsorb specific miRNAs to regulate the expression of their target gene mRNAs. The conclusions are: i) differentially expressed circRNAs had corresponding miRNA binding sites. These circRNAs regulated the expression of target genes through interactions with miRNAs and might become new molecular biomarkers for GC in the future. ii) Differentially expressed genes may be involved in the occurrence of GC via a variety of mechanisms. iii) CD44, CXXC5, MYH9, MALAT1 and other genes may have important implications for the occurrence and development of GC through the regulation, interaction, and mutual influence of circRNA-miRNA-mRNA via different mechanisms.

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

PubMed Central

2014-01-01

Background Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e.g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community. PMID:24456189

Radiation Fibrosis of the Vocal Fold: From Man to Mouse

PubMed Central

Johns, Michael M.; Kolachala, Vasantha; Berg, Eric; Muller, Susan; Creighton, Frances X.; Branski, Ryan C.

2013-01-01

Objectives To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis to ultimately develop both treatment and prevention paradigms. Design Translational study using archived human and fresh murine irradiated vocal fold tissue. Methods 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis were identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray as well as real-time transcriptional analyses was performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis. Results Human irradiated vocal folds demonstrated increased collagen transcription with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-β increased in the lamina propria. Conclusion Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability that occurs in patients following laryngeal irradiation. Irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis. PMID:23242839

Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma.

PubMed

Serra, Pierre; Petat, Arthur; Maury, Jean-Michel; Thivolet-Bejui, Françoise; Chalabreysse, Lara; Barritault, Marc; Ebran, Nathalie; Milano, Gérard; Girard, Nicolas; Brevet, Marie

2018-04-01

The systematic assessment of anti-programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in lung adenocarcinomas is becoming standard practice. However, the assessment of PD-L1 expression on small tissue specimens needs to be evaluated and the association with other features more thoroughly analyzed. This retrospective single center study evaluated the immunohistochemical expression of the SP263 anti-PD-L1 antibody on tissue microarrays (TMA) of 152 surgically resected lung adenocarcinomas, using a 25% positivity threshold. The positive cases and 50 randomly chosen negative cases in tissue microarray (TMA) were reassessed on whole tissue sections. The results were correlated to clinical, histopathological and to molecular data obtained through the screening of 214 mutations in 26 genes (LungCarta panel, Agena Biosciences). Among 152 primary lung adenocarcinomas, 19 cases (13%) showed PD-L1 expression. The agreement between TMA and whole tissue sections was 89%, specificity was 97%. PD-L1 expression was correlated to RAS mutations (p = .04), RAS/TP53 co-mutations (p = .01) and to the solid or acinar subtype (p = .048). With the SP263 PD-L1 antibody, small samples appear as a reliable means to evaluate the PD-L1 status in lung adenocarcinoma. The association between PD-L1 expression and RAS/TP53 mutations may have clinical relevance to predict the efficacy of PD-1/PD-L1 immune checkpoints inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

PubMed Central

Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

2012-01-01

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. PMID:23209738

Genes that characterize T3-predominant Graves' thyroid tissues.

PubMed

Matsumoto, Chisa; Ito, Mitsuru; Yamada, Hiroya; Yamakawa, Noriko; Yoshida, Hiroshi; Date, Arisa; Watanabe, Mikio; Hidaka, Yoh; Iwatani, Yoshinori; Miyauchi, Akira; Takano, Toru

2013-02-01

3,5,3'-Triiodothyronine (T(3))-predominant Graves' disease is characterized by the increasing volume of thyroid goiter resulting in poor prognosis. Although type 1 and type 2 iodothyronine deiodinases (DIO1 and DIO2 respectively) are known to be overexpressed in the thyroid tissues of T(3)-predominant Graves' disease, the pathogenesis of this disease is still unclear. The aim of our study is to identify genes that characterize T(3)-predominant Graves' disease tissue in order to clarify the molecular mechanism of this disease. mRNAs from two thyroid tissues of both typical T(3)-predominant and common-type Graves' disease were analyzed with DNA microarrays with probes for 28 869 genes. Genes identified to be differentially expressed between the two groups were further analyzed in the second and third screenings using 70 Graves' thyroid tissues by real-time quantitative RT-PCR. Twenty-three candidate genes were selected as being differentially expressed in the first screening with microarrays. Among these, seven genes, leucine-rich repeat neuronal 1 (LRRN1), bone morphogenetic protein 8a (BMP8A), N-cadherin (CDH2), phosphodiesterase 1A (PDE1A), creatine kinase mitochondrial 2 (CKMT2), integrin beta-3 (ITGB3), and protein tyrosine phosphatase non-receptor type 4 (PTPN4), were confirmed to be differentially expressed in DIO1 or DIO2 over- and underexpressing Graves' tissues. These genes are related to the characteristics of T(3)-predominant Graves' disease, such as high titer level of serum anti-TSH receptor antibody, high free T(3) to free thyroxine ratio, and a large goiter size. They might play a role in the pathogenesis of T(3)-predominant Graves' disease.

Two-way learning with one-way supervision for gene expression data.

PubMed

Wong, Monica H T; Mutch, David M; McNicholas, Paul D

2017-03-04

A family of parsimonious Gaussian mixture models for the biclustering of gene expression data is introduced. Biclustering is accommodated by adopting a mixture of factor analyzers model with a binary, row-stochastic factor loadings matrix. This particular form of factor loadings matrix results in a block-diagonal covariance matrix, which is a useful property in gene expression analyses, specifically in biomarker discovery scenarios where blood can potentially act as a surrogate tissue for other less accessible tissues. Prior knowledge of the factor loadings matrix is useful in this application and is reflected in the one-way supervised nature of the algorithm. Additionally, the factor loadings matrix can be assumed to be constant across all components because of the relationship desired between the various types of tissue samples. Parameter estimates are obtained through a variant of the expectation-maximization algorithm and the best-fitting model is selected using the Bayesian information criterion. The family of models is demonstrated using simulated data and two real microarray data sets. The first real data set is from a rat study that investigated the influence of diabetes on gene expression in different tissues. The second real data set is from a human transcriptomics study that focused on blood and immune tissues. The microarray data sets illustrate the biclustering family's performance in biomarker discovery involving peripheral blood as surrogate biopsy material. The simulation studies indicate that the algorithm identifies the correct biclusters, most optimally when the number of observation clusters is known. Moreover, the biclustering algorithm identified biclusters comprised of biologically meaningful data related to insulin resistance and immune function in the rat and human real data sets, respectively. Initial results using real data show that this biclustering technique provides a novel approach for biomarker discovery by enabling blood to be used as a surrogate for hard-to-obtain tissues.

Characterization of genetic variability of Venezuelan equine encephalitis viruses

DOE PAGES

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.; ...

2016-04-07

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Circular RNA Expression Profile of Pancreatic Ductal Adenocarcinoma Revealed by Microarray.

PubMed

Li, Haimin; Hao, Xiaokun; Wang, Huimin; Liu, Zhengcai; He, Yong; Pu, Meng; Zhang, Hongtao; Yu, Hengchao; Duan, Juanli; Qu, Shibin

2016-01-01

Circular RNAs (circRNAs) are a special novel type of a stable, diverse and conserved noncoding RNA in mammalian cells. Particularly in cancer, circRNAs have been reported to be widely involved in the physiological/pathological process of life. However, it is unclear whether circRNAs are specifically involved in pancreatic ductal adenocarcinoma (PDAC). We investigated the expression profile of circRNAs in six PDAC cancer samples and paired adjacent normal tissues using microarray. A high-throughput circRNA microarray was used to identify dysregulated circular RNAs in six PDAC patients. Bioinformatic analyses were applied to study these differentially expressed circRNAs. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to confirm these results. We revealed and confirmed that a number of circRNAs were dysregulated, which suggests a potential role in pancreatic cancer. this study demonstrates that clusters of circRNAs are aberrantly expressed in PDAC compared with normal samples and provides new potential targets for the future treatment of PDAC and novel insights into PDAC biology. © 2016 The Author(s) Published by S. Karger AG, Basel.

Loss of Smad4 in colorectal cancer induces resistance to 5-fluorouracil through activating Akt pathway

PubMed Central

Zhang, B; Zhang, B; Chen, X; Bae, S; Singh, K; Washington, M K; Datta, P K

2014-01-01

Background: Higher frequency of Smad4 inactivation or loss of expression is observed in metastasis of colorectal cancer (CRC) leading to unfavourable survival and contributes to chemoresistance. However, the molecular mechanism of how Smad4 regulates chemosensitivity of CRC is unknown. Methods: We evaluated how the loss of Smad4 in CRC enhanced chemoresistance to 5-fluorouracil (5-FU) using two CRC cell lines in vitro and in vivo. Immunoblotting with cell and tumour lysates and immunohistochemical analyses with tissue microarray were performed. Results: Knockdown or loss of Smad4 induced tumorigenicity, migration, invasion, angiogenesis, metastasis, and 5-FU resistance. Smad4 expression in mouse tumours regulated cell-cycle regulatory proteins leading to Rb phosphorylation. Loss of Smad4 activated Akt pathway that resulted in upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-w, and Survivin. Suppression of phosphatidylinositol-3-kinase (PI3K)/Akt pathway by LY294002 restored chemosensitivity of Smad4-deficient cells to 5-FU. Vascular endothelial growth factor-induced angiogenesis in Smad4-deficient cells might also lead to chemoresistance. Low levels of Smad4 expression in CRC tissues correlated with higher levels of Bcl-2 and Bcl-w and with poor overall survival as observed in immunohistochemical staining of tissue microarrays. Conclusion: Loss of Smad4 in CRC patients induces resistance to 5-FU-based therapy through activation of Akt pathway and inhibitors of this pathway may sensitise these patients to 5-FU. PMID:24384683

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP

PubMed Central

Coutinho, Rita; Clear, Andrew J.; Mazzola, Emanuele; Owen, Andrew; Greaves, Paul; Wilson, Andrew; Matthews, Janet; Lee, Abigail; Alvarez, Rute; da Silva, Maria Gomes; Cabeçadas, José; Neuberg, Donna; Calaminici, Maria; Gribben, John G.

2015-01-01

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma. PMID:25425693

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP.

PubMed

Coutinho, Rita; Clear, Andrew J; Mazzola, Emanuele; Owen, Andrew; Greaves, Paul; Wilson, Andrew; Matthews, Janet; Lee, Abigail; Alvarez, Rute; da Silva, Maria Gomes; Cabeçadas, José; Neuberg, Donna; Calaminici, Maria; Gribben, John G

2015-03-01

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma. Copyright© Ferrata Storti Foundation.

Doxycycline affects gene expression profiles in aortic tissues in a rat model of vascular calcification.

PubMed

Lu, Hailin; Jiang, Wenhong; Yang, Han; Qin, Zhong; Guo, Si-En; Hu, Ming; Qin, Xiao

2017-11-01

Vitamin D 3 -induced vascular calcification (VC) in rats shares many phenotypical similarities with calcification occurring in human atherosclerosis, diabetes mellitus and chronic kidney disease, thereby it is a reliable model for identifying chemopreventive agents. Doxycycline has been shown to effectively attenuated VC. This study aimed to explore the effects of doxycycline on gene expression profiles in VC rats. The model of VC in rats was established by subcutaneous injection of vitamin D3 for 3days. Doxycycline at 120mgkg -1 day -1 was given via subcutaneous injection for 14days. Rat pathological changes, calcium deposition and calcium content in aortic tissues were measured by Hematoxylin-eosin, von Kossa staining and colorimetry, respectively. The gene change profile of aortic tissues after doxycycline treatment was assessed by Gene Microarray analysis using the Agilent Whole Rat Genome Oligo Microarray. The results showed that doxycycline significantly decreased the deposition of calcium, reduced the relative calcification area and alleviated pathological injury in aortic tissues. In addition, doxycycline treatment altered 88 gene expressions compared with untreated VD group. Of these, 61 genes were down-regulated and 27 genes were up-regulated. The functions of differentially expressed (DE) genes were involved in neutrophil chemotaxis, chronic inflammatory response, negative regulation of apoptotic process, cellular response to mechanical stimulus and immune response, etc. In conclusions, this study might provide the potential novel insights into the molecular mechanisms of doxycycline on VC. Copyright © 2017. Published by Elsevier Inc.

The Role of the Immune Response in the Pathogenesis of Thyroid Eye Disease: A Reassessment

PubMed Central

Rosenbaum, James T.; Choi, Dongseok; Wong, Amanda; Wilson, David J.; Grossniklaus, Hans E.; Harrington, Christina A.; Dailey, Roger A.; Ng, John D.; Steele, Eric A.; Czyz, Craig N.; Foster, Jill A.; Tse, David; Alabiad, Chris; Dubovy, Sander; Parekh, Prashant K.; Harris, Gerald J.; Kazim, Michael; Patel, Payal J.; White, Valerie A.; Dolman, Peter J.; Edward, Deepak P.; Alkatan, Hind M.; al Hussain, Hailah; Selva, Dinesh; Yeatts, R. Patrick; Korn, Bobby S.; Kikkawa, Don O.; Stauffer, Patrick; Planck, Stephen R.

2015-01-01

Background Although thyroid eye disease is a common complication of Graves’ disease, the pathogenesis of the orbital disease is poorly understood. Most authorities implicate the immune response as an important causal factor. We sought to clarify pathogenesis by using gene expression microarray. Methods An international consortium of ocular pathologists and orbital surgeons contributed formalin fixed orbital biopsies. RNA was extracted from orbital tissue from 20 healthy controls, 25 patients with thyroid eye disease (TED), 25 patients with nonspecific orbital inflammation (NSOI), 7 patients with sarcoidosis and 6 patients with granulomatosis with polyangiitis (GPA). Tissue was divided into a discovery set and a validation set. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays which include 54,000 probe sets. Results Principal component analysis showed that gene expression from tissue from patients with TED more closely resembled gene expression from healthy control tissue in comparison to gene expression characteristic of sarcoidosis, NSOI, or granulomatosis with polyangiitis. Unsupervised cluster dendrograms further indicated the similarity between TED and healthy controls. Heat maps based on gene expression for cytokines, chemokines, or their receptors showed that these inflammatory markers were associated with NSOI, sarcoidosis, or GPA much more frequently than with TED. Conclusion This is the first study to compare gene expression in TED to gene expression associated with other causes of exophthalmos. The juxtaposition shows that inflammatory markers are far less characteristic of TED relative to other orbital inflammatory diseases. PMID:26371757

Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

PubMed Central

Chen, YuXian; Shen, Jun; Lu, HuaDing; Zeng, Chun; Ren, JianHua; Zeng, Hua; Li, ZhiFu; Chen, ShaoMing; Cai, DaoZhang; Zhao, Qing

2012-01-01

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA. PMID:22384228

Microarray analysis of subcutaneous adipose tissue from mature cows with divergent body weight gain after feed restriction and realimentation

USDA-ARS?s Scientific Manuscript database

Body weight response to periods of feed restriction and realimentation is critical and relevant to the agricultural industry. The purpose of this study was to evaluate differentially expressed genes identified in subcutaneous adipose tissue collected from cows divergent in body weight (BW) gain afte...

Prognostic value of matrix metalloproteinase 9 expression in patients with juvenile nasopharyngeal angiofibroma: tissue microarray analysis.

PubMed

Sun, Xicai; Guo, Limin; Wang, Jingjing; Wang, Huan; Liu, Zhuofu; Liu, Juan; Yu, Huapeng; Hu, Li; Li, Han; Wang, Dehui

2014-08-01

Although JNA is a benign neoplasm histopathologically, it has a propensity for locally destructive growth and remains a higher postoperative recurrence rate. The aim of this study was to analyze the expression and localization of MMP-9 in JNA using tissue microarray to elucidate its correlation with clinicopathological features and recurrence. The expression of MMP-9 was assessed by immunohistochemistry in a tissue microarray from 70 patients with JNA and 10 control subjects. Correlation between the levels of MMP-9 expression and clinicopathologic variables, as well as tumor recurrence, were analyzed. MMP-9 was detected in perivascular and extravascular less differentiated cells and stromal cells of patients with JNA but not in the matured vascular endothelial cells of these patients. The presence of MMP-9 expression in JNA was correlated with patient's age (p=0.001). Spearman correlation analysis suggested that high expression of MMP-9 in JNA had negative correlation with patient's age (r=-0.412, p<0.001). The recurrence rate in JNA patients with high MMP-9 expression was significantly higher than those with low MMP-9 expression (p=0.002). In multivariate and ROC curve analysis, MMP-9 was a good prognostic factor for tumor recurrence of JNA. Higher MMP-9 expression is a poor prognostic factor for patients with JNA who have been surgically treated. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes specifically overexpressed in tumor epithelia.

PubMed

Badea, Liviu; Herlea, Vlad; Dima, Simona Olimpia; Dumitrascu, Traian; Popescu, Irinel

2008-01-01

The precise details of pancreatic ductal adenocarcinoma (PDAC) pathogenesis are still insufficiently known, requiring the use of high-throughput methods. However, PDAC is especially difficult to study using microarrays due to its strong desmoplastic reaction, which involves a hyperproliferating stroma that effectively "masks" the contribution of the minoritary neoplastic epithelial cells. Thus it is not clear which of the genes that have been found differentially expressed between normal and whole tumor tissues are due to the tumor epithelia and which simply reflect the differences in cellular composition. To address this problem, laser microdissection studies have been performed, but these have to deal with much smaller tissue sample quantities and therefore have significantly higher experimental noise. In this paper we combine our own large sample whole-tissue study with a previously published smaller sample microdissection study by Grützmann et al. to identify the genes that are specifically overexpressed in PDAC tumor epithelia. The overlap of this list of genes with other microarray studies of pancreatic cancer as well as with the published literature is impressive. Moreover, we find a number of genes whose over-expression appears to be inversely correlated with patient survival: keratin 7, laminin gamma 2, stratifin, platelet phosphofructokinase, annexin A2, MAP4K4 and OACT2 (MBOAT2), which are all specifically upregulated in the neoplastic epithelia, rather than the tumor stroma. We improve on other microarray studies of PDAC by putting together the higher statistical power due to a larger number of samples with information about cell-type specific expression and patient survival.

EST resources and establishment and validation of a 16k cDNA microarray from Atlantic cod (Gadus morhua).

PubMed

Edvardsen, Rolf B; Malde, Ketil; Mittelholzer, Christian; Taranger, Geir Lasse; Nilsen, Frank

2011-03-01

The Atlantic cod, Gadus morhua, is an important species both for traditional fishery and increasingly also in fish farming. The Atlantic cod is also under potential threat from various environmental changes such as pollution and climate change, but the biological impact of such changes are not well known, in particular when it comes to sublethal effects that can be difficult to assert. Modern molecular and genomic approaches have revolutionized biological research during the last decade, and offer new avenues to study biological functions and e.g. the impact of anthropogenic activities at different life-stages for a given organism. In order to develop genomic data and genomic tools for Atlantic cod we conducted a program were we constructed 20 cDNA libraries, and produced and analyzed 44006 expressed sequence tags (ESTs) from these. Several tissues are represented in the multiple cDNA libraries, that differ in either sexual maturation or immulogical stimulation. This approach allowed us to identify genes that are expressed in particular tissues, life-stages or in response to specific stimuli, and also gives us information about potential functions of the transcripts. The ESTs were used to construct a 16k cDNA microarray to further investigate the cod transcriptome. Microarray analyses were preformed on pylorus, pituitary gland, spleen and testis of sexually maturing male cod. The four different tissues displayed tissue specific transcriptomes demonstrating that the cDNA array is working as expected and will prove to be a powerful tool in further experiments. Copyright © 2010 Elsevier Inc. All rights reserved.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

PubMed

Redmond, Latasha C; Pang, Christopher J; Dumur, Catherine; Haar, Jack L; Lloyd, Joyce A

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

PubMed Central

Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

Microarray profiling of human white adipose tissue after exogenous leptin injection.

PubMed

Taleb, S; Van Haaften, R; Henegar, C; Hukshorn, C; Cancello, R; Pelloux, V; Hanczar, B; Viguerie, N; Langin, D; Evelo, C; Zucker, J; Clément, K; Saris, W H M

2006-03-01

Leptin is a secreted adipocyte hormone that plays a key role in the regulation of body weight homeostasis. The leptin effect on human white adipose tissue (WAT) is still debated. The aim of this study was to assess whether the administration of polyethylene glycol-leptin (PEG-OB) in a single supraphysiological dose has transcriptional effects on genes of WAT and to identify its target genes and functional pathways in WAT. Blood samples and WAT biopsies were obtained from 10 healthy nonobese men before treatment and 72 h after the PEG-OB injection, leading to an approximate 809-fold increase in circulating leptin. The WAT gene expression profile before and after the PEG-OB injection was compared using pangenomic microarrays. Functional gene annotations based on the gene ontology of the PEG-OB regulated genes were performed using both an 'in house' automated procedure and GenMAPP (Gene Microarray Pathway Profiler), designed for viewing and analyzing gene expression data in the context of biological pathways. Statistical analysis of microarray data revealed that PEG-OB had a major down-regulated effect on WAT gene expression, as we obtained 1,822 and 100 down- and up-regulated genes, respectively. Microarray data were validated using reverse transcription quantitative PCR. Functional gene annotations of PEG-OB regulated genes revealed that the functional class related to immunity and inflammation was among the most mobilized PEG-OB pathway in WAT. These genes are mainly expressed in the cell of the stroma vascular fraction in comparison with adipocytes. Our observations support the hypothesis that leptin could act on WAT, particularly on genes related to inflammation and immunity, which may suggest a novel leptin target pathway in human WAT.

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses.

PubMed

Glaros, Trevor G; Blancett, Candace D; Bell, Todd M; Natesan, Mohan; Ulrich, Robert G

2015-01-01

The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

PubMed Central

Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

2005-01-01

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

ARACNe-based inference, using curated microarray data, of Arabidopsis thaliana root transcriptional regulatory networks

PubMed Central

2014-01-01

Background Uncovering the complex transcriptional regulatory networks (TRNs) that underlie plant and animal development remains a challenge. However, a vast amount of data from public microarray experiments is available, which can be subject to inference algorithms in order to recover reliable TRN architectures. Results In this study we present a simple bioinformatics methodology that uses public, carefully curated microarray data and the mutual information algorithm ARACNe in order to obtain a database of transcriptional interactions. We used data from Arabidopsis thaliana root samples to show that the transcriptional regulatory networks derived from this database successfully recover previously identified root transcriptional modules and to propose new transcription factors for the SHORT ROOT/SCARECROW and PLETHORA pathways. We further show that these networks are a powerful tool to integrate and analyze high-throughput expression data, as exemplified by our analysis of a SHORT ROOT induction time-course microarray dataset, and are a reliable source for the prediction of novel root gene functions. In particular, we used our database to predict novel genes involved in root secondary cell-wall synthesis and identified the MADS-box TF XAL1/AGL12 as an unexpected participant in this process. Conclusions This study demonstrates that network inference using carefully curated microarray data yields reliable TRN architectures. In contrast to previous efforts to obtain root TRNs, that have focused on particular functional modules or tissues, our root transcriptional interactions provide an overview of the transcriptional pathways present in Arabidopsis thaliana roots and will likely yield a plethora of novel hypotheses to be tested experimentally. PMID:24739361

Microarray evaluation of gene expression profiles in inflamed and healthy human dental pulp: the role of IL1beta and CD40 in pulp inflammation.

PubMed

Gatta, V; Zizzari, V L; Dd ' Amico, V; Salini, L; D' Aurora, M; Franchi, S; Antonucci, I; Sberna, M T; Gherlone, E; Stuppia, L; Tetè, S

2012-01-01

Dental pulp undergoes a number of changes passing from healthy status to inflammation due to deep decay. These changes are regulated by several genes resulting differently expressed in inflamed and healthy dental pulp, and the knowledge of the processes underlying this differential expression is of great relevance in the identification of the pathogenesis of the disease. In this study, the gene expression profile of inflamed and healthy dental pulps were compared by microarray analysis, and data obtained were analyzed by Ingenuity Pathway Analysis (IPA) software. This analysis allows to focus on a variety of genes, typically expressed in inflamed tissues. The comparison analysis showed an increased expression of several genes in inflamed pulp, among which IL1β and CD40 resulted of particular interest. These results indicate that gene expression profile of human dental pulp in different physiological and pathological conditions may become an useful tool for improving our knowledge about processes regulating pulp inflammation.

Database construction for PromoterCAD: synthetic promoter design for mammals and plants.

PubMed

Nishikata, Koro; Cox, Robert Sidney; Shimoyama, Sayoko; Yoshida, Yuko; Matsui, Minami; Makita, Yuko; Toyoda, Tetsuro

2014-03-21

Synthetic promoters can control a gene's timing, location, and expression level. The PromoterCAD web server ( http://promotercad.org ) allows the design of synthetic promoters to control plant gene expression, by novel arrangement of cis-regulatory elements. Recently, we have expanded PromoterCAD's scope with additional plant and animal data: (1) PLACE (Plant Cis-acting Regulatory DNA Elements), including various sized sequence motifs; (2) PEDB (Mammalian Promoter/Enhancer Database), including gene expression data for mammalian tissues. The plant PromoterCAD data now contains 22 000 Arabidopsis thaliana genes, 2 200 000 microarray measurements in 20 growth conditions and 79 tissue organs and developmental stages, while the new mammalian PromoterCAD data contains 679 Mus musculus genes and 65 000 microarray measurements in 96 tissue organs and cell types ( http://promotercad.org/mammal/ ). This work presents step-by-step instructions for adding both regulatory motif and gene expression data to PromoterCAD, to illustrate how users can expand PromoterCAD functionality for their own applications and organisms.

ROKU: a novel method for identification of tissue-specific genes

PubMed Central

Kadota, Koji; Ye, Jiazhen; Nakai, Yuji; Terada, Tohru; Shimizu, Kentaro

2006-01-01

Background One of the important goals of microarray research is the identification of genes whose expression is considerably higher or lower in some tissues than in others. We would like to have ways of identifying such tissue-specific genes. Results We describe a method, ROKU, which selects tissue-specific patterns from gene expression data for many tissues and thousands of genes. ROKU ranks genes according to their overall tissue specificity using Shannon entropy and detects tissues specific to each gene if any exist using an outlier detection method. We evaluated the capacity for the detection of various specific expression patterns using synthetic and real data. We observed that ROKU was superior to a conventional entropy-based method in its ability to rank genes according to overall tissue specificity and to detect genes whose expression pattern are specific only to objective tissues. Conclusion ROKU is useful for the detection of various tissue-specific expression patterns. The framework is also directly applicable to the selection of diagnostic markers for molecular classification of multiple classes. PMID:16764735

Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

PubMed Central

2014-01-01

Background KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. Methods We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. Results KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Conclusions Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it may represent a novel target for biomarker development and a novel therapeutic target for breast cancer. PMID:24628760

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.

PubMed

Couture, Camille; Zaniolo, Karine; Carrier, Patrick; Lake, Jennifer; Patenaude, Julien; Germain, Lucie; Guérin, Sylvain L

2016-02-01

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protein profiles associated with survival in lung adenocarcinoma

PubMed Central

Chen, Guoan; Gharib, Tarek G; Wang, Hong; Huang, Chiang-Ching; Kuick, Rork; Thomas, Dafydd G.; Shedden, Kerby A.; Misek, David E.; Taylor, Jeremy M. G.; Giordano, Thomas J.; Kardia, Sharon L. R.; Iannettoni, Mark D.; Yee, John; Hogg, Philip J.; Orringer, Mark B.; Hanash, Samir M.; Beer, David G.

2003-01-01

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer. PMID:14573703

Image microarrays (IMA): Digital pathology's missing tool

PubMed Central

Hipp, Jason; Cheng, Jerome; Pantanowitz, Liron; Hewitt, Stephen; Yagi, Yukako; Monaco, James; Madabhushi, Anant; Rodriguez-canales, Jaime; Hanson, Jeffrey; Roy-Chowdhuri, Sinchita; Filie, Armando C.; Feldman, Michael D.; Tomaszewski, John E.; Shih, Natalie NC.; Brodsky, Victor; Giaccone, Giuseppe; Emmert-Buck, Michael R.; Balis, Ulysses J.

2011-01-01

Introduction: The increasing availability of whole slide imaging (WSI) data sets (digital slides) from glass slides offers new opportunities for the development of computer-aided diagnostic (CAD) algorithms. With the all-digital pathology workflow that these data sets will enable in the near future, literally millions of digital slides will be generated and stored. Consequently, the field in general and pathologists, specifically, will need tools to help extract actionable information from this new and vast collective repository. Methods: To address this limitation, we designed and implemented a tool (dCORE) to enable the systematic capture of image tiles with constrained size and resolution that contain desired histopathologic features. Results: In this communication, we describe a user-friendly tool that will enable pathologists to mine digital slides archives to create image microarrays (IMAs). IMAs are to digital slides as tissue microarrays (TMAs) are to cell blocks. Thus, a single digital slide could be transformed into an array of hundreds to thousands of high quality digital images, with each containing key diagnostic morphologies and appropriate controls. Current manual digital image cut-and-paste methods that allow for the creation of a grid of images (such as an IMA) of matching resolutions are tedious. Conclusion: The ability to create IMAs representing hundreds to thousands of vetted morphologic features has numerous applications in education, proficiency testing, consensus case review, and research. Lastly, in a manner analogous to the way conventional TMA technology has significantly accelerated in situ studies of tissue specimens use of IMAs has similar potential to significantly accelerate CAD algorithm development. PMID:22200030

Prostate Cancer Pathology Resource Network

DTIC Science & Technology

2012-07-01

microarrays (TMAs), serum, plasma , buffy coat, prostatic fluid, and derived specimens (DNA and RNA); these specimens are linked to clinical and...research community. The specimens in the PCBN include tissues from prostatectomies, serum, plasma , buffy coat, prostatic fluid, derived specimens such...prostatectomy, seminal vesicles), body fluids (serum, plasma , buffy coat, prostatic fluid; most can be matched to tumor and benign tissue), and

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

PubMed

Tiwari, Jagesh Kumar; Devi, Sapna; Sundaresha, S; Chandel, Poonam; Ali, Nilofer; Singh, Brajesh; Bhardwaj, Vinay; Singh, Bir Pal

2015-06-01

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization.

PubMed

Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

2011-12-07

It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A-bomb radiation may affect the increased amount of CNA as a hallmark of GIN and, subsequently, be associated with a higher histologic grade in breast cancer found in A-bomb survivors.

Microarray analysis of port wine stains before and after pulsed dye laser treatment.

PubMed

Laquer, Vivian T; Hevezi, Peter A; Albrecht, Huguette; Chen, Tina S; Zlotnik, Albert; Kelly, Kristen M

2013-02-01

Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were able to identify genes that may participate in formation of PWS and PDL effects. Our preliminary results indicate changes in gene expression of angiogenesis-related genes, suggesting that dysregulation of angiogenic signals and/or components may contribute to PWS pathology. Copyright © 2012 Wiley Periodicals, Inc.

The clinicopathologic association of c-MET overexpression in Iranian gastric carcinomas; an immunohistochemical study of tissue microarrays

PubMed Central

2012-01-01

Background c-MET is an oncogene protein that plays important role in gastric carcinogenesis and has been introduced as a prognostic marker and potential therapeutic target. The aim of this study was to evaluate the frequency of c-MET overexpression and its relationship with clinicopathological variables in gastric cancer of Iranian population using tissue microarray. Methods In a cross sectional study, representative paraffin blocks of 130 patients with gastric carcinoma treated by curative gastrectomy during a 2 years period of 2008–2009 in two university hospitals in Tehran-Iran were collected in tissue microarray and c-MET expression was studied by immunohistochemical staining. Results Finally 124 cases were evaluated, constituted of 99 male and 25 female with the average age of 61.5 years. In 71% (88/124) of tumors, c-MET high expression was found. c-MET high expression was more associated with intestinal than diffuse tumor type (P = 0.04), deeper tumor invasion, pT3 and pT4 versus pT1 and pT2 (P = 0.014), neural invasion (P = 0.002) and advanced TNM staging, stage 3 and 4 versus stage 1 and2 (P = 0.044). The c-MET high expression was not associated with age, sex, tumor location, differentiation grade and distant metastasis, but relative associations with lymph node metastasis (P = 0.065) and vascular invasion (P = 0.078) were observed. Conclusions c-MET oncogene protein was frequently overexpressed in Iranian gastric carcinomas and it was related to clinicopathological characteristics such as tumor type, depth of invasion, neural invasion and TNM staging. It can also support the idea that c-MET is a potential marker for target therapy in Iranian gastric cancer. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9744598757151429 PMID:22640970

Association of tumor-infiltrating T-cell density with molecular subtype, racial ancestry and clinical outcomes in prostate cancer.

PubMed

Kaur, Harsimar B; Guedes, Liana B; Lu, Jiayun; Maldonado, Laneisha; Reitz, Logan; Barber, John R; De Marzo, Angelo M; Tosoian, Jeffrey J; Tomlins, Scott A; Schaeffer, Edward M; Joshu, Corinne E; Sfanos, Karen S; Lotan, Tamara L

2018-05-30

The inflammatory microenvironment plays an important role in the pathogenesis and progression of tumors and may be associated with somatic genomic alterations. We examined the association of tumor-infiltrating T-cell density with clinical-pathologic variables, tumor molecular subtype, and oncologic outcomes in surgically treated primary prostate cancer occurring in patients of European-American or African-American ancestry. We evaluated 312 primary prostate tumors, enriched for patients with African-American ancestry and high grade disease. Tissue microarrays were immunostained for CD3, CD8, and FOXP3 and were previously immunostained for ERG and PTEN using genetically validated protocols. Image analysis for quantification of T-cell density in tissue microarray tumor spots was performed. Automated quantification of T-cell densities in tumor-containing regions of tissue microarray spots and standard histologic sections were correlated (r = 0.73, p < 0.00001) and there was good agreement between visual and automated T-cell density counts on tissue microarray spots (r = 0.93, p < 0.00001). There was a significant correlation between CD3+, CD8+, and FOXP3+ T-cell densities (p < 0.00001), but these were not associated with most clinical or pathologic variables. Increased T-cell density was significantly associated with ERG positivity (median 309 vs. 188 CD3+ T cells/mm 2 ; p = 0.0004) and also with PTEN loss (median 317 vs. 192 CD3+ T cells/mm 2 ; p = 0.001) in the combined cohort of matched European-American and African-American ancestry patients. The same association or a similar trend was present in patients of both ancestries when analyzed separately. When the African-American patients from the matched race set were combined with a separate high grade set of African-American cases, there was a weak association of increased FOXP3+ T-cell densities with increased risk of metastasis in multivariable analysis. Though high T-cell density is associated with specific molecular subclasses of prostate cancer, we did not find an association of T-cell density with racial ancestry.

Downregulation of TES by hypermethylation in glioblastoma reduces cell apoptosis and predicts poor clinical outcome.

PubMed

Bai, Yu; Zhang, Quan-Geng; Wang, Xin-Hua

2014-12-11

Gliomas are the most common human brain tumors. Glioblastoma, also known as glioblastoma multiform (GBM), is the most aggressive, malignant, and lethal glioma. The investigation of prognostic and diagnostic molecular biomarkers in glioma patients to provide direction on clinical practice is urgent. Recent studies demonstrated that abnormal DNA methylation states play a key role in the pathogenesis of this kind of tumor. In this study, we want to identify a novel biomarker related to glioma initiation and find the role of the glioma-related gene. We performed a methylation-specific microarray on the promoter region to identify methylation gene(s) that may affect outcome of GBM patients. Normal and GBM tissues were collected from Tiantan Hospital. Genomic DNA was extracted from these tissues and analyzed with a DNA promoter methylation microarray. Testis derived transcript (TES) protein expression was analyzed by immunohistochemistry in paraffin-embedded patient tissues. Western blotting was used to detect TES protein expression in the GBM cell line U251 with or without 5-aza-dC treatment. Cell apoptosis was evaluated by flow cytometry analysis using Annexin V/PI staining. We found that the TES promoter was hypermethylated in GBM compared to normal brain tissues under DNA promoter methylation microarray analysis. The GBM patients with TES hypermethylation had a short overall survival (P <0.05, log-rank test). Among GBM samples, reduced TES protein level was detected in 33 (89.2%) of 37 tumor tissues by immunohistochemical staining. Down regulation of TES was also correlated with worse patient outcome (P <0.05, log-rank test). Treatment on the GBM cell line U251 with 5-aza-dC can greatly increase TES expression, confirming the hypermethylation of TES promoter in GBM. Up-regulation of TES prompts U251 apoptosis significantly. This study demonstrated that both TES promoter hypermethylation and down-regulated protein expression significantly correlated with worse patient outcome. Treatment on the GBM cell line (U251) with 5-aza-dC can highly release TES expression resulting in significant apoptosis in these cells. Our findings suggest that the TES gene is a novel tumor suppressor gene and might represent a valuable prognostic marker for glioblastoma, indicating a potential target for future GBM therapy.

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shakoor, N; Nair, R; Crasta, O

2014-01-23

Background: Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results: This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specificmore » probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e. g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions: Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.« less

MicroRNA-301a-3p promotes pancreatic cancer progression via negative regulation of SMAD4

PubMed Central

Zhang, Kundong; Cen, Gang; Jiang, Tao; Cao, Jun; Huang, Kejian; Zhao, Qian; Qiu, Zhengjun

2015-01-01

Background Aim to determine the clinicopathological and prognostic role of miR-301a-3p in pancreatic ductal adenocarcinoma(PDAC), to investigate the biological mechanism of miR-301a-3p in vitro and in vivo. Methods By tissue microarray analysis, we studied miR-301a-3p expression in PDAC patients and its clinicopathological correlations as well as prognostic significance. qRT-PCR was used to test miR-301a-3p expression in PDAC tissues and cell lines. Functional experiments including in vitro and in vivo were performed. Results Significantly higher expression of miR-301a-3p were found in PDAC patients with lymph node metastasis and advanced pathological stages and identified as an independent prognostic factor for worse survival. In PDAC samples and cell lines, miR-301a-3p was significantly up-regulated compared with matched non-tumor tissues and normal pancreatic ductal cells, respectively. Overexpression of miR-301a-3p enhanced PDAC cells colony, invasion and migration abilities in vitro as well as tumorigenicity in vivo. Furthermore, SMAD4 was identified as a target gene of miR-301a-3p by cell as well as mice xenograft experiments. In PDAC tissue microarray, a significantly inverse correlation between miR-301a-3p ISH scores and SMAD4 IHC scores were observed in both tumor and corresponding non-tumor tissues. Conclusion MiR-301a-3p functions as a novel oncogene in PDAC and the oncogenic activity may involve its inhibition of the target gene SMAD4. PMID:26019136

High SPDEF May Identify Patients Who Will Have a Prolonged Response to Androgen Deprivation Therapy

PubMed Central

Haller, Andrew C.; Tan, Wei; Payne-Ondracek, Rochelle; Underwood, Willie; Tian, Lili; Morrison, Carl; Li, Fengzhi

2015-01-01

Background Due to the indolent nature of prostate cancer, new prognostic measures are needed to identify patients with life threatening disease. SAM pointed domain-containing Ets transcription factor (SPDEF) has been associated with good prognosis and demonstrates an intimate relationship with the androgen receptor (AR), however its role in prostate cancer progression remains unclear. Methods A tissue microarray constructed from cores of 713 consecutive radical prostatectomy specimens were immunohistochemically stained for SPDEF and correlated with progression free and metastatic free survival. In vitro studies assessed growth rate, migration, and sensitivity to bicalutamide to explore mechanisms behind the tissue microarray observations. Results Patients with high SPDEF demonstrate longer metastases free survival after receiving the standard of care (HR = 9.80, P = 0.006). SPDEF expression corresponded with bicalutamide growth inhibition and apoptosis induction in all cell lines studied. In addition, a feed-forward loop of AR-SPEF expression regulation is observed. Conclusions SPDEF may be clinically useful to identify patients who will have extended benefits from androgen deprivation therapy. In vitro observations suggest SPDEF mediates initial sensitivity to androgen deprivation therapy through both AR regulation and downstream events. PMID:24375440

Loss of Cytoplasmic CDK1 Predicts Poor Survival in Human Lung Cancer and Confers Chemotherapeutic Resistance

PubMed Central

Zhang, Chunyu; Elkahloun, Abdel G.; Robertson, Matthew; Gills, Joell J.; Tsurutani, Junji; Shih, Joanna H.; Fukuoka, Junya; Hollander, M. Christine; Harris, Curtis C.; Travis, William D.; Jen, Jin; Dennis, Phillip A.

2011-01-01

The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. PMID:21887332

High resolution time course analysis of gene expression from the liver and pituitary

PubMed Central

Hughes, Michael E.; DiTacchio, Luciano; Hayes, Kevin; Pullivarthy, Sandhya R.; Panda, Satchidananda; Hogenesch, John

2009-01-01

In both the suprachiasmatic nucleus and peripheral tissues, the circadian oscillator drives rhythmic transcription of downstream target genes. Recently, a number of studies have used DNA microarrays to systematically identify oscillating transcripts in plants, fruit flies, rats and mice. These studies have identified several dozen to many hundred rhythmically expressed genes by sampling tissues every four hours for one, two, or more days. To extend this work, we have performed DNA microarray analysis on RNA derived from the mouse pituitary sampled every hour for two days. COSOPT and Fisher's G-test were employed at a false-discovery rate less than 5% to identify more than 250 genes in the pituitary that oscillate with a 24-hour period length. We found that increasing the frequency of sampling across the circadian day dramatically increased the statistical power of both COSOPT and Fisher's G-test, resulting in considerably more high-confidence identifications of rhythmic transcripts than previously described. Finally, to extend the utility of these data sets, a web-based resource has been constructed at http://wasabi.itmat.upenn.edu/circa/mouse that is freely available to the research community. PMID:18419295

Tissue Microarray Analysis Applied to Bone Diagenesis

PubMed Central

Mello, Rafael Barrios; Silva, Maria Regina Regis; Alves, Maria Teresa Seixas; Evison, Martin Paul; Guimarães, Marco Aurelio; Francisco, Rafaella Arrabaca; Astolphi, Rafael Dias; Iwamura, Edna Sadayo Miazato

2017-01-01

Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered. PMID:28051148

Unconventional microarray design reveals the response to obesity is largely tissue specific: analysis of common and divergent responses to diet-induced obesity in insulin-sensitive tissues.

PubMed

Lee, Robyn K; Hittel, Dustin S; Nyamandi, Vongai Z; Kang, Li; Soh, Jung; Sensen, Christoph W; Shearer, Jane

2012-04-01

Obesity is a chronic condition involving the excessive accumulation of adipose tissue that adversely affects all systems in the body. The aim of the present study was to employ an unbiased, genome-wide assessment of transcript abundance in order to identify common gene expression pathways within insulin-sensitive tissues in response to dietary-induced diabetes. Following 20 weeks of chow or high-fat feeding (60% kcal), age-matched mice underwent a euglycemic-hyperinsulinemic clamp to assess insulin sensitivity. High-fat-fed animals were obese and highly insulin resistant, disposing of ∼75% less glucose compared with their chow-fed counterparts. Tissues were collected, and gene expression was examined by microarray in 4 tissues known to exhibit obesity-related metabolic disturbances: white adipose tissue, skeletal muscle, liver, and heart. A total of 463 genes were differentially expressed between diets. Analysis of individual tissues showed skeletal muscle to exhibit the largest number of differentially expressed genes (191) in response to high-fat feeding, followed by adipose tissue (169), liver (115), and heart (65). Analyses revealed that the response of individual genes to obesity is distinct and largely tissue specific, with less than 10% of transcripts being shared among tissues. Although transcripts are largely tissue specific, a systems approach shows numerous commonly activated pathways, including those involved in signal transduction, inflammation, oxidative stress, substrate transport, and metabolism. This suggests a coordinated attempt by tissues to limit metabolic perturbations occurring in early-stage obesity. Many identified genes were associated with a variety of disorders, thereby serving as potential links between obesity and its related health risks.

Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

PubMed Central

Pichler, Martin; Zatloukal, Kurt

2013-01-01

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples. PMID:23936242

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PubMed

Wang, Xin-xin; Ma, Yue; Li, Qing; Jiang, Bao-qi; Lan, Jing

2012-10-01

To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results.

PubMed

Dai, Yilin; Guo, Ling; Li, Meng; Chen, Yi-Bu

2012-06-08

Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

Microarray expression profiles of genes in lung tissues of rats subjected to focal cerebral ischemia-induced lung injury following bone marrow-derived mesenchymal stem cell transplantation.

PubMed

Hu, Yue; Xiong, Liu-Lin; Zhang, Piao; Wang, Ting-Hua

2017-01-01

Ischemia-induced stroke is the most common disease of the nervous system and is associated with a high mortality rate worldwide. Cerebral ischemia may lead to remote organ dysfunction, particular in the lungs, resulting in lung injury. Nowadays, bone marrow-derived mesenchymal stem cells (BMSCs) are widely studied in clinical trials as they may provide an effective solution to the treatment of neurological and cardiac diseases; however, the underlying molecular mechanisms remain unknown. In this study, a model of permanent focal cerebral ischemia-induced lung injury was successfully established and confirmed by neurological evaluation and lung injury scores. We demonstrated that the transplantation of BMSCs (passage 3) via the tail vein into the lung tissues attenuated lung injury. In order to elucidate the underlying molecular mechanisms, we analyzed the gene expression profiles in lung tissues from the rats with focal cerebral ischemia and transplanted with BMSCs using a Gene microarray. Moreover, the Gene Ontology database was employed to determine gene function. We found that the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) were downregulated in the BMSC transplantation groups, compared with the control group. These results suggested that BMSC transplantation may attenuate lung injury following focal cerebral ischemia and that this effect is associated with the downregulation of TGF-β, PDGF and the PI3K-AKT pathway.

High expression of AFAP1-AS1 is associated with poor survival and short-term recurrence in pancreatic ductal adenocarcinoma.

PubMed

Ye, Yibiao; Chen, Jie; Zhou, Yu; Fu, Zhiqiang; Zhou, Quanbo; Wang, YingXue; Gao, Wenchao; Zheng, ShangYou; Zhao, Xiaohui; Chen, Tao; Chen, Rufu

2015-04-30

Pancreatic ductal adenocarcinoma (PDAC) is still a lethal malignancy. Long noncoding RNAs (lncRNAs) have been shown to play a critical role in cancer development and progression. Here we identified overexpression of the lncRNA AFAP1-AS1 in PDAC patients and evaluated its prognostic and functional relevance. The global lncRNA expression profile in PDAC was measured by lncRNA microarray. Expression of AFAP1-AS1 was evaluated by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) in 90 PDAC tissue samples and adjacent normal tissues. The impact of AFAP1-AS1 expression on cell proliferation, migration, and invasion were evaluated in vitro using knockdown and ectopic expression strategies. Microarray analysis revealed that up-regulation of AFAP1-AS1 expression in PDAC tissues compared with normal adjacent tissues, which was confirmed by RT-qPCR in 69/90 cases (76.7%). Its overexpression was associated with lymph node metastasis, perineural invasion, and poor survival. When using AFAP1-AS1 as a prognostic marker, the areas under ROC curves were 0.8669 and 0.9370 for predicting tumor progression within 6 months and 1 year, respectively. In vitro functional experiments involving knockdown of AFAP1-AS1 resulted in attenuated PDAC cell proliferation, migration, and invasion. Ectopic expression of AFAP1-AS1 promoted cell proliferation, migration, and invasion. AFAP1-AS1 is a potential novel prognostic marker to predict the clinical outcome of PDAC patients after surgery and may be a rational target for therapy.

Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells

PubMed Central

Chen, Michael Q.; Xie, Xiaoyan; Wilson, Kitchener D.; Sun, Ning; Wu, Joseph C.; Giovangrandi, Laurent; Kovacs, Gregory T. A.

2010-01-01

Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 µA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in β-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation. PMID:20652088

A prototype for unsupervised analysis of tissue microarrays for cancer research and diagnostics.

PubMed

Chen, Wenjin; Reiss, Michael; Foran, David J

2004-06-01

The tissue microarray (TMA) technique enables researchers to extract small cylinders of tissue from histological sections and arrange them in a matrix configuration on a recipient paraffin block such that hundreds can be analyzed simultaneously. TMA offers several advantages over traditional specimen preparation by maximizing limited tissue resources and providing a highly efficient means for visualizing molecular targets. By enabling researchers to reliably determine the protein expression profile for specific types of cancer, it may be possible to elucidate the mechanism by which healthy tissues are transformed into malignancies. Currently, the primary methods used to evaluate arrays involve the interactive review of TMA samples while they are viewed under a microscope, subjectively evaluated, and scored by a technician. This process is extremely slow, tedious, and prone to error. In order to facilitate large-scale, multi-institutional studies, a more automated and reliable means for analyzing TMAs is needed. We report here a web-based prototype which features automated imaging, registration, and distributed archiving of TMAs in multiuser network environments. The system utilizes a principal color decomposition approach to identify and characterize the predominant staining signatures of specimens in color space. This strategy was shown to be reliable for detecting and quantifying the immunohistochemical expression levels for TMAs.

Feasibility of using tissue microarray cores of paraffin-embedded breast cancer tissue for measurement of gene expression: a proof-of-concept study.

PubMed

Drury, Suzanne; Salter, Janine; Baehner, Frederick L; Shak, Steven; Dowsett, Mitch

2010-06-01

To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. Four matched 0.6 mm cores of invasive breast tumour and two 10 microm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan assays were performed on the 21 genes of the Oncotype DX Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. RNA yield was lower from 0.6 mm cores than from 10 microm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies.

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

PubMed Central

Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

2007-01-01

Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that Actichip is a powerful alternative to commercial high density microarrays for cytoskeleton gene profiling in normal or pathological samples. Actichip is available upon request. PMID:17727702

Integrating Colon Cancer Microarray Data: Associating Locus-Specific Methylation Groups to Gene Expression-Based Classifications.

PubMed

Barat, Ana; Ruskin, Heather J; Byrne, Annette T; Prehn, Jochen H M

2015-11-23

Recently, considerable attention has been paid to gene expression-based classifications of colorectal cancers (CRC) and their association with patient prognosis. In addition to changes in gene expression, abnormal DNA-methylation is known to play an important role in cancer onset and development, and colon cancer is no exception to this rule. Large-scale technologies, such as methylation microarray assays and specific sequencing of methylated DNA, have been used to determine whole genome profiles of CpG island methylation in tissue samples. In this article, publicly available microarray-based gene expression and methylation data sets are used to characterize expression subtypes with respect to locus-specific methylation. A major objective was to determine whether integration of these data types improves previously characterized subtypes, or provides evidence for additional subtypes. We used unsupervised clustering techniques to determine methylation-based subgroups, which are subsequently annotated with three published expression-based classifications, comprising from three to six subtypes. Our results showed that, while methylation profiles provide a further basis for segregation of certain (Inflammatory and Goblet-like) finer-grained expression-based subtypes, they also suggest that other finer-grained subtypes are not distinctive and can be considered as a single subtype.

Integrating Colon Cancer Microarray Data: Associating Locus-Specific Methylation Groups to Gene Expression-Based Classifications

PubMed Central

Barat, Ana; Ruskin, Heather J.; Byrne, Annette T.; Prehn, Jochen H. M.

2015-01-01

Recently, considerable attention has been paid to gene expression-based classifications of colorectal cancers (CRC) and their association with patient prognosis. In addition to changes in gene expression, abnormal DNA-methylation is known to play an important role in cancer onset and development, and colon cancer is no exception to this rule. Large-scale technologies, such as methylation microarray assays and specific sequencing of methylated DNA, have been used to determine whole genome profiles of CpG island methylation in tissue samples. In this article, publicly available microarray-based gene expression and methylation data sets are used to characterize expression subtypes with respect to locus-specific methylation. A major objective was to determine whether integration of these data types improves previously characterized subtypes, or provides evidence for additional subtypes. We used unsupervised clustering techniques to determine methylation-based subgroups, which are subsequently annotated with three published expression-based classifications, comprising from three to six subtypes. Our results showed that, while methylation profiles provide a further basis for segregation of certain (Inflammatory and Goblet-like) finer-grained expression-based subtypes, they also suggest that other finer-grained subtypes are not distinctive and can be considered as a single subtype. PMID:27600244

Comparing transformation methods for DNA microarray data

PubMed Central

Thygesen, Helene H; Zwinderman, Aeilko H

2004-01-01

Background When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include subtraction of an estimated background signal, subtracting the reference signal, smoothing (to account for nonlinear measurement effects), and more. Different authors use different approaches, and it is generally not clear to users which method they should prefer. Results We used the ratio between biological variance and measurement variance (which is an F-like statistic) as a quality measure for transformation methods, and we demonstrate a method for maximizing that variance ratio on real data. We explore a number of transformations issues, including Box-Cox transformation, baseline shift, partial subtraction of the log-reference signal and smoothing. It appears that the optimal choice of parameters for the transformation methods depends on the data. Further, the behavior of the variance ratio, under the null hypothesis of zero biological variance, appears to depend on the choice of parameters. Conclusions The use of replicates in microarray experiments is important. Adjustment for the null-hypothesis behavior of the variance ratio is critical to the selection of transformation method. PMID:15202953

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Biomarkers of Selenium Action in Prostate Cancer

DTIC Science & Technology

2006-03-01

without BPH) transition zone tissue of a 42-year-old man ac- cording to previously described methods [4]. The pre- sence of contaminating epithelial...protein secreted by cells using a sensitive ELISA method . Replicating the conditions used for the microarray analyses, cells were fed fresh medium...4 Introduction Biomarkers of selenium actions in prostate tissue would be of great value in stratifying patients

Status of epigenetic chromatin modification enzymes and esophageal squamous cell carcinoma risk in northeast Indian population.

PubMed

Singh, Virendra; Singh, Laishram C; Singh, Avninder P; Sharma, Jagannath; Borthakur, Bibhuti B; Debnath, Arundhati; Rai, Avdhesh K; Phukan, Rup K; Mahanta, Jagadish; Kataki, Amal C; Kapur, Sujala; Saxena, Sunita

2015-01-01

Esophageal cancer incidence is reported in high frequency in northeast India. The etiology is different from other population at India due to wide variations in dietary habits or nutritional factors, tobacco/betel quid chewing and alcohol habits. Since DNA methylation, histone modification and miRNA-mediated epigenetic processes alter the gene expression, the involvement of these processes might be useful to find out epigenetic markers of esophageal cancer risk in northeast Indian population. The present investigation was aimed to carryout differential expression profiling of chromatin modification enzymes in tumor and normal tissue collected from esophageal squamous cell carcinoma (ESCC) patients. Differential mRNA expression profiling and their validation was done by quantitative real time PCR and tissue microarray respectively. Univariate and multiple logistic regression analysis were used to analyze the epidemiological data. mRNA expression data was analyzed by Student t-test. Fisher exact test was used for tissue microarray data analysis. Higher expression of enzymes regulating methylation (DOT1L and PRMT1) and acetylation (KAT7, KAT8, KAT2A and KAT6A) of histone was found associated with ESCC risk. Tissue microarray done in independent cohort of 75 patients revealed higher nuclear protein expression of KAT8 and PRMT1 in tumor similar to mRNA expression. Expression status of PRMT1 and KAT8 was found declined as we move from low grade to high grade tumor. Betel nut chewing, alcohol drinking and dried fish intake were significantly associated with increased risk of esophageal cancer among the study subject. Study suggests the association of PRMT1 and KAT8 with esophageal cancer risk and its involvement in the transition process of low to high grade tumor formation. The study exposes the differential status of chromatin modification enzymes between tumor and normal tissue and points out that relaxed state of chromatin facilitates more transcriptionally active genome in esophageal carcinogenesis.

Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Fei; Maslov, Sergei; Yoo, Shinjae

Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

DOE PAGES

He, Fei; Maslov, Sergei; Yoo, Shinjae; ...

2016-05-25

Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

Characterization of human septic sera induced gene expression modulation in human myocytes

PubMed Central

Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

2009-01-01

To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. PMID:19684886

Designing oligo libraries taking alternative splicing into account

NASA Astrophysics Data System (ADS)

Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

2001-06-01

We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

Src kinases in chondrosarcoma chemoresistance and migration: dasatinib sensitises to doxorubicin in TP53 mutant cells

PubMed Central

van Oosterwijk, J G; van Ruler, M A J H; Briaire-de Bruijn, I H; Herpers, B; Gelderblom, H; van de Water, B; Bovée, J V M G

2013-01-01

Background: Chondrosarcomas are malignant cartilage-forming tumours of bone. Because of their resistance to conventional chemotherapy and radiotherapy, currently no treatment strategies exist for unresectable and metastatic chondrosarcoma. Previously, PI3K/AKT/GSK3β and Src kinase pathways were shown to be activated in chondrosarcoma cell lines. Our aim was to investigate the role of these kinases in chemoresistance and migration in chondrosarcoma in relation to TP53 mutation status. Methods: We used five conventional and three dedifferentiated chondrosarcoma cell lines and investigated the effect of PI3K/AKT/GSK3β pathway inhibition (enzastaurin) and Src pathway inhibition (dasatinib) in chemoresistance using WST assay and live cell imaging with AnnexinV staining. Immunohistochemistry on tissue microarrays (TMAs) containing 157 cartilaginous tumours was performed for Src family members. Migration assays were performed with the RTCA xCelligence System. Results: Src inhibition was found to overcome chemoresistance, to induce apoptosis and to inhibit migration. Cell lines with TP53 mutations responded better to combination therapy than wild-type cell lines (P=0.002). Tissue microarray immunohistochemistry confirmed active Src (pSrc) signalling, with Fyn being most abundantly expressed (76.1%). Conclusion: These results strongly indicate Src family kinases, in particular Fyn, as a potential target for the treatment of inoperable and metastatic chondrosarcomas, and to sensitise for doxorubicin especially in the presence of TP53 mutations. PMID:23922104

Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells

PubMed Central

Liao, Yunfei; Chen, Lulu; Feng, Yong; Shen, Jacson; Gao, Yan; Cote, Gregory; Choy, Edwin; Harmon, David; Mankin, Henry; Hornicek, Francis; Duan, Zhenfeng

2017-01-01

Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed on tumor cells that suppresses the T cell-mediated immune response. Therapies targeting the PD-L1 pathway promote anti-tumor immunity and have shown promising results in some types of cancers. However, the functional and therapeutic roles of PD-L1 in osteosarcoma remain largely unknown. In this study, we found that PD-L1 protein was expressed in osteosarcoma cell lines and tissue microarray of patient tumors. Tissue microarray immunohistochemistry analysis showed that the overall and five-year survival rates of patients with high levels of PD-L1 expression were significantly shorter than patients with low levels. High levels of PD-L1 expression were also associated with metastasis in osteosarcoma patients. Furthermore, we applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to target PD-L1 gene at the DNA level in osteosarcoma cell lines. We found that the expression of PD-L1 could be efficiently disrupted by CRISPR/Cas9 system and PD-L1 knockdown increased drug sensitivities for doxorubicin and paclitaxel. These results suggest that PD-L1 is an independent prognostic factor in osteosarcoma and that PD-L1 knockout by CRISPR/Cas9 may be a therapeutic approach for the treatment of osteosarcoma. PMID:28415820

Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells.

PubMed

Liao, Yunfei; Chen, Lulu; Feng, Yong; Shen, Jacson; Gao, Yan; Cote, Gregory; Choy, Edwin; Harmon, David; Mankin, Henry; Hornicek, Francis; Duan, Zhenfeng

2017-05-02

Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed on tumor cells that suppresses the T cell-mediated immune response. Therapies targeting the PD-L1 pathway promote anti-tumor immunity and have shown promising results in some types of cancers. However, the functional and therapeutic roles of PD-L1 in osteosarcoma remain largely unknown. In this study, we found that PD-L1 protein was expressed in osteosarcoma cell lines and tissue microarray of patient tumors. Tissue microarray immunohistochemistry analysis showed that the overall and five-year survival rates of patients with high levels of PD-L1 expression were significantly shorter than patients with low levels. High levels of PD-L1 expression were also associated with metastasis in osteosarcoma patients. Furthermore, we applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to target PD-L1 gene at the DNA level in osteosarcoma cell lines. We found that the expression of PD-L1 could be efficiently disrupted by CRISPR/Cas9 system and PD-L1 knockdown increased drug sensitivities for doxorubicin and paclitaxel. These results suggest that PD-L1 is an independent prognostic factor in osteosarcoma and that PD-L1 knockout by CRISPR/Cas9 may be a therapeutic approach for the treatment of osteosarcoma.

[Expression of cell adhesion molecules in acute leukemia cell].

PubMed

Ju, Xiaoping; Peng, Min; Xu, Xiaoping; Lu, Shuqing; Li, Yao; Ying, Kang; Xie, Yi; Mao, Yumin; Xia, Fang

2002-11-01

To investigate the role of cell adhesion molecule in the development and extramedullary infiltration (EI) of acute leukemia. The expressions of neural cell adhesion molecule (NCAM) gene, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) genes in 25 acute leukemia patients bone marrow cells were detected by microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). The expressions of NCAM, ICAM-1 and VCAM-1 gene were significantly higher in acute leukemia cells and leukemia cells with EI than in normal tissues and leukemia cells without EI, respectively, both by cDNA microarray and by RT-PCR. The cDNA microarray is a powerful technique in analysis of acute leukemia cells associated genes. High expressions of cell adhesion molecule genes might be correlated with leukemia pathogenesis and infiltration of acute leukemia cell.

Deep learning based tissue analysis predicts outcome in colorectal cancer.

PubMed

Bychkov, Dmitrii; Linder, Nina; Turkki, Riku; Nordling, Stig; Kovanen, Panu E; Verrill, Clare; Walliander, Margarita; Lundin, Mikael; Haglund, Caj; Lundin, Johan

2018-02-21

Image-based machine learning and deep learning in particular has recently shown expert-level accuracy in medical image classification. In this study, we combine convolutional and recurrent architectures to train a deep network to predict colorectal cancer outcome based on images of tumour tissue samples. The novelty of our approach is that we directly predict patient outcome, without any intermediate tissue classification. We evaluate a set of digitized haematoxylin-eosin-stained tumour tissue microarray (TMA) samples from 420 colorectal cancer patients with clinicopathological and outcome data available. The results show that deep learning-based outcome prediction with only small tissue areas as input outperforms (hazard ratio 2.3; CI 95% 1.79-3.03; AUC 0.69) visual histological assessment performed by human experts on both TMA spot (HR 1.67; CI 95% 1.28-2.19; AUC 0.58) and whole-slide level (HR 1.65; CI 95% 1.30-2.15; AUC 0.57) in the stratification into low- and high-risk patients. Our results suggest that state-of-the-art deep learning techniques can extract more prognostic information from the tissue morphology of colorectal cancer than an experienced human observer.

Differential Expression of MicroRNA and Predicted Targets in Pulmonary Sarcoidosis

PubMed Central

Crouser, Elliott D.; Julian, Mark W.; Crawford, Melissa; Shao, Guohong; Yu, Lianbo; Planck, Stephen R.; Rosenbaum, James T.; Nana-Sinkam, S. Patrick

2014-01-01

Background Recent studies show that various inflammatory diseases are regulated at the level of RNA translation by small non-coding RNAs, termed microRNAs (miRNAs). We sought to determine whether sarcoidosis tissues harbor a distinct pattern of miRNA expression and then considered their potential molecular targets. Methods and Results Genome-wide microarray analysis of miRNA expression in lung tissue and peripheral blood mononuclear cells (PBMCs) was performed and differentially expressed (DE)-miRNAs were then validated by real-time PCR. A distinct pattern of DE-miRNA expression was identified in both lung tissue and PBMCs of sarcoidosis patients. A subgroup of DE-miRNAs common to lung and lymph node tissues were predicted to target transforming growth factor (TGFβ)-regulated pathways. Likewise, the DE-miRNAs identified in PBMCs of sarcoidosis patients were predicted to target the TGFβ-regulated “wingless and integrase-1” (WNT) pathway. Conclusions This study is the first to profile miRNAs in sarcoidosis tissues and to consider their possible roles in disease pathogenesis. Our results suggest that miRNA regulate TGFβ and related WNT pathways in sarcoidosis tissues, pathways previously incriminated in the pathogenesis of sarcoidosis. PMID:22209793

Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

PubMed

Villeneuve, L; Wang, Rong-Lin; Bencic, David C; Biales, Adam D; Martinović, Dalma; Lazorchak, James M; Toth, Gregory; Ankley, Gerald T

2009-08-01

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stemmer, Kerstin; Ellinger-Ziegelbauer, Heidrun; Lotz, Kerstin

2006-11-15

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treatedmore » for 7 days and 6 months with 10 and 1 mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H and E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230{sub 2}.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.« less

Dynamics of 17alpha-ethynylestradiol exposure in rainbow trout (Oncorhynchus mykiss): absorption, tissue distribution, and hepatic gene expression pattern.

PubMed

Skillman, Ann D; Nagler, James J; Hook, Sharon E; Small, Jack A; Schultz, Irvin R

2006-11-01

17alpha-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor alpha (ERalpha) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ERalpha mRNA, and EE2 were measured using enzyme-linked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography-mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ERalpha mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ERalpha) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive.

DYNAMICS OF 17α-ETHYNYLESTRADIOL EXPOSURE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS): ABSORPTION, TISSUE DISTRIBUTION, AND HEPATIC GENE EXPRESSION PATTERN

PubMed Central

Skillman, Ann D.; Nagler, James J.; Hook, Sharon E.; Small, Jack A.; Schultz, Irvin R.

2008-01-01

17α-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor α (ERα) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ERα mRNA, and EE2 were measured using enzyme-linked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography–mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ERα mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ERα) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive. PMID:17089724

Tissue-enriched expression profiles in Aedes aegypti identify hemocyte-specific transcriptome responses to infection

PubMed Central

Choi, Young-Jun; Fuchs, Jeremy F.; Mayhew, George F.; Yu, Helen E.; Christensen, Bruce M.

2012-01-01

Hemocytes are integral components of mosquito immune mechanisms such as phagocytosis, melanization, and production of antimicrobial peptides. However, our understanding of hemocyte-specific molecular processes and their contribution to shaping the host immune response remains limited. To better understand the immunophysiological features distinctive of hemocytes, we conducted genome-wide analysis of hemocyte-enriched transcripts, and examined how tissue-enriched expression patterns change with the immune status of the host. Our microarray data indicate that the hemocyte-enriched trascriptome is dynamic and context-dependent. Analysis of transcripts enriched after bacterial challenge in circulating hemocytes with respect to carcass added a dimension to evaluating infection-responsive genes and immune-related gene families. We resolved patterns of transcriptional change unique to hemocytes from those that are likely shared by other immune responsive tissues, and identified clusters of genes preferentially induced in hemocytes, likely reflecting their involvement in cell type specific functions. In addition, the study revealed conserved hemocyte-enriched molecular repertoires which might be implicated in core hemocyte function by cross-species meta-analysis of microarray expression data from Anopheles gambiae and Drosophila melanogaster. PMID:22796331

SYMBIOmatics: Synergies in Medical Informatics and Bioinformatics – exploring current scientific literature for emerging topics

PubMed Central

Rebholz-Schuhman, Dietrich; Cameron, Graham; Clark, Dominic; van Mulligen, Erik; Coatrieux, Jean-Louis; Del Hoyo Barbolla, Eva; Martin-Sanchez, Fernando; Milanesi, Luciano; Porro, Ivan; Beltrame, Francesco; Tollis, Ioannis; Van der Lei, Johan

2007-01-01

Background The SYMBIOmatics Specific Support Action (SSA) is "an information gathering and dissemination activity" that seeks "to identify synergies between the bioinformatics and the medical informatics" domain to improve collaborative progress between both domains (ref. to ). As part of the project experts in both research fields will be identified and approached through a survey. To provide input to the survey, the scientific literature was analysed to extract topics relevant to both medical informatics and bioinformatics. Results This paper presents results of a systematic analysis of the scientific literature from medical informatics research and bioinformatics research. In the analysis pairs of words (bigrams) from the leading bioinformatics and medical informatics journals have been used as indication of existing and emerging technologies and topics over the period 2000–2005 ("recent") and 1990–1990 ("past"). We identified emerging topics that were equally important to bioinformatics and medical informatics in recent years such as microarray experiments, ontologies, open source, text mining and support vector machines. Emerging topics that evolved only in bioinformatics were system biology, protein interaction networks and statistical methods for microarray analyses, whereas emerging topics in medical informatics were grid technology and tissue microarrays. Conclusion We conclude that although both fields have their own specific domains of interest, they share common technological developments that tend to be initiated by new developments in biotechnology and computer science. PMID:17430562

Mapping of oxidative stress responses of human tumor cells following photodynamic therapy using hexaminolevulinate

PubMed Central

Cekaite, Lina; Peng, Qian; Reiner, Andrew; Shahzidi, Susan; Tveito, Siri; Furre, Ingegerd E; Hovig, Eivind

2007-01-01

Background Photodynamic therapy (PDT) involves systemic or topical administration of a lesion-localizing photosensitizer or its precursor, followed by irradiation of visible light to cause singlet oxygen-induced damage to the affected tissue. A number of mechanisms seem to be involved in the protective responses to PDT, including activation of transcription factors, heat shock proteins, antioxidant enzymes and apoptotic pathways. Results In this study, we address the effects of a destructive/lethal hexaminolevulinate (HAL) mediated PDT dose on the transcriptome by using transcriptional exon evidence oligo microarrays. Here, we confirm deviations in the steady state expression levels of previously identified early defence response genes and extend this to include unreported PDT inducible gene groups, most notably the metallothioneins and histones. HAL-PDT mediated stress also altered expression of genes encoded by mitochondrial DNA (mtDNA). Further, we report PDT stress induced alternative splicing. Specifically, the ATF3 alternative isoform (deltaZip2) was up-regulated, while the full-length variant was not changed by the treatment. Results were independently verified by two different technological microarray platforms. Good microarray, RT-PCR and Western immunoblotting correlation for selected genes support these findings. Conclusion Here, we report new insights into how destructive/lethal PDT alters the transcriptome not only at the transcriptional level but also at post-transcriptional level via alternative splicing. PMID:17692132

Gene expression profiling of two distinct neuronal populations in the rodent spinal cord.

PubMed

Ryge, Jesper; Westerdahl, Ann-Charlotte; Alstrøm, Preben; Kiehn, Ole

2008-01-01

In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord.

Gene Expression Profiling of Two Distinct Neuronal Populations in the Rodent Spinal Cord

PubMed Central

Alstrøm, Preben; Kiehn, Ole

2008-01-01

Background In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. Methodology/Principal Findings We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50–250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. Conclusions/Significance We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord. PMID:18923679

Early induction of cytokines/cytokine receptors and Cox2, and activation of NF-κB in 4-nitroquinoline 1-oxide-induced murine oral cancer model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yu-Ching; Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan; Ho, Heng-Chien

2012-07-15

The purpose of this study was to identify the genes induced early in murine oral carcinogenesis. Murine tongue tumors induced by the carcinogen, 4-nitroquinoline 1-oxide (4-NQO), and paired non-tumor tissues were subjected to microarray analysis. Hierarchical clustering of upregulated genes in the tumor tissues revealed an association of induced genes with inflammation. Cytokines/cytokine receptors induced early were subsequently identified, clearly indicating their involvement in oral carcinogenesis. Hierarchical clustering also showed that cytokine-mediated inflammation was possibly linked with Mapk6. Cox2 exhibited the greatest extent (9–18 fold) of induction in the microarray data, and its early induction was observed in a 2more » h painting experiment by RT-PCR. MetaCore analysis showed that overexpressed Cox2 may interact with p53 and transcriptionally inhibit expression of several downstream genes. A painting experiment in transgenic mice also demonstrated that NF-κB activates early independently of Cox2 induction. MetaCore analysis revealed the most striking metabolic alterations in tumor tissues, especially in lipid metabolism resulting from the reduction of Pparα and Rxrg. Reduced expression of Mapk12 was noted, and MetaCore analysis established its relationship with decreased efficiency of Pparα phosphorylation. In conclusion, in addition to cytokines/cytokine receptors, the early induction of Cox2 and NF-κB activation is involved in murine oral carcinogenesis.« less

Loss of Smad4 in colorectal cancer induces resistance to 5-fluorouracil through activating Akt pathway.

PubMed

Zhang, B; Zhang, B; Chen, X; Bae, S; Singh, K; Washington, M K; Datta, P K

2014-02-18

Higher frequency of Smad4 inactivation or loss of expression is observed in metastasis of colorectal cancer (CRC) leading to unfavourable survival and contributes to chemoresistance. However, the molecular mechanism of how Smad4 regulates chemosensitivity of CRC is unknown. We evaluated how the loss of Smad4 in CRC enhanced chemoresistance to 5-fluorouracil (5-FU) using two CRC cell lines in vitro and in vivo. Immunoblotting with cell and tumour lysates and immunohistochemical analyses with tissue microarray were performed. Knockdown or loss of Smad4 induced tumorigenicity, migration, invasion, angiogenesis, metastasis, and 5-FU resistance. Smad4 expression in mouse tumours regulated cell-cycle regulatory proteins leading to Rb phosphorylation. Loss of Smad4 activated Akt pathway that resulted in upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-w, and Survivin. Suppression of phosphatidylinositol-3-kinase (PI3K)/Akt pathway by LY294002 restored chemosensitivity of Smad4-deficient cells to 5-FU. Vascular endothelial growth factor-induced angiogenesis in Smad4-deficient cells might also lead to chemoresistance. Low levels of Smad4 expression in CRC tissues correlated with higher levels of Bcl-2 and Bcl-w and with poor overall survival as observed in immunohistochemical staining of tissue microarrays. Loss of Smad4 in CRC patients induces resistance to 5-FU-based therapy through activation of Akt pathway and inhibitors of this pathway may sensitise these patients to 5-FU.

Role of Macrophage-Induced Inflammation in Mesothelioma

DTIC Science & Technology

2010-07-01

in human mesothelioma tumors and correlate immune cell infiltration with histopathologic subtype (months 1-6). Using tumor tissue microarrays of... histopathologic subtype (months 1-6). • Acquired 71 fixed and paraffin-embedded mesothelioma tumor samples • Prepared mesothelioma tumor tissue...Biol., 2008. 84: p. 1-8. 5. Dave, S.S., et al., Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating

Tissue microarrays and digital image analysis.

PubMed

Ryan, Denise; Mulrane, Laoighse; Rexhepaj, Elton; Gallagher, William M

2011-01-01

Tissue microarrays (TMAs) have recently emerged as very valuable tools for high-throughput pathological assessment, especially in the cancer research arena. This important technology, however, has yet to fully penetrate into the area of toxicology. Here, we describe the creation of TMAs representative of samples produced from conventional toxicology studies within a large-scale, multi-institutional pan-European project, PredTox. PredTox, short for Predictive Toxicology, formed part of an EU FP6 Integrated Project, Innovative Medicines for Europe (InnoMed), and aimed to study pre-clinically 16 compounds of known liver and/or kidney toxicity. In more detail, TMAs were constructed from materials corresponding to the full face sections of liver and kidney from rats treated with different drug candidates by members of the consortium. We also describe the process of digital slide scanning of kidney and liver sections, in the context of creating an online resource of histopathological data.

A DNA microarray for identification of selected Korean birds based on mitochondrial cytochrome c oxidase I gene sequences.

PubMed

Chung, In-Hyuk; Yoo, Hye Sook; Eah, Jae-Yong; Yoon, Hyun-Kyu; Jung, Jin-Wook; Hwang, Seung Yong; Kim, Chang-Bae

2010-10-01

DNA barcoding with the gene encoding cytochrome c oxidase I (COI) in the mitochondrial genome has been proposed as a standard marker to identify and discover animal species. Some migratory wild birds are suspected of transmitting avian influenza and pose a threat to aircraft safety because of bird strikes. We have previously reported the COI gene sequences of 92 Korean bird species. In the present study, we developed a DNA microarray to identify 17 selected bird species on the basis of nucleotide diversity. We designed and synthesized 19 specific oligonucleotide probes; these probes were arrayed on a silylated glass slide. The length of the probes was 19-24 bps. The COI sequences amplified from the tissues of the selected birds were labeled with a fluorescent probe for microarray hybridization, and unique hybridization patterns were detected for each selected species. These patterns may be considered diagnostic patterns for species identification. This microarray system will provide a sensitive and a high-throughput method for identification of Korean birds.

Elucidation of the effect of brain cortex tetrapeptide Cortagen on gene expression in mouse heart by microarray.

PubMed

Anisimov, Sergey V; Khavinson, Vladimir Kh; Anisimov, Vladimir N

2004-01-01

Aging is associated with significant alterations in gene expression in numerous organs and tissues. Anti-aging therapy with peptide bioregulators holds much promise for the correction of age-associated changes, making a screening for their molecular targets in tissues an important question of modern gerontology. The synthetic tetrapeptide Cortagen (Ala-Glu-Asp-Pro) was obtained by directed synthesis based on amino acid analysis of natural brain cortex peptide preparation Cortexin. In humans, Cortagen demonstrated a pronounced therapeutic effect upon the structural and functional posttraumatic recovery of peripheral nerve tissue. Importantly, other effects were also observed in cardiovascular and cerebrovascular parameters. Based on these latter observations, we hypothesized that acute course of Cortagen treatment, large-scale transcriptome analysis, and identification of transcripts with altered expression in heart would facilitate our understanding of the mechanisms responsible for this peptide biological effects. We therefore analyzed the expression of 15,247 transcripts in the heart of female 6-months CBA mice receiving injections of Cortagen for 5 consecutive days was studied by cDNA microarrays. Comparative analysis of cDNA microarray hybridisation with heart samples from control and experimental group revealed 234 clones (1,53% of the total number of clones) with significant changes of expression that matched 110 known genes belonging to various functional categories. Maximum up- and down-regulation was +5.42 and -2.86, respectively. Intercomparison of changes in cardiac expression profile induced by synthetic peptides (Cortagen, Vilon, Epitalon) and pineal peptide hormone melatonin revealed both common and specific effects of Cortagen upon gene expression in heart.

Comprehensive Genetic Analysis of Early Host Body Reactions to the Bioactive and Bio-Inert Porous Scaffolds

PubMed Central

Ehashi, Tomo; Takemura, Taro; Hanagata, Nobutaka; Minowa, Takashi; Kobayashi, Hisatoshi; Ishihara, Kazuhiko; Yamaoka, Tetsuji

2014-01-01

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1β and IL-10 are important cytokines in tissue responses to biomaterials because IL-1β promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1β was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations. PMID:24454803

Potential upstream regulators of cannabinoid receptor 1 signaling in prostate cancer: a Bayesian network analysis of data from a tissue microarray.

PubMed

Häggström, Jenny; Cipriano, Mariateresa; Forshell, Linus Plym; Persson, Emma; Hammarsten, Peter; Stella, Nephi; Fowler, Christopher J

2014-08-01

The endocannabinoid system regulates cancer cell proliferation, and in prostate cancer a high cannabinoid CB1 receptor expression is associated with a poor prognosis. Down-stream mediators of CB1 receptor signaling in prostate cancer are known, but information on potential upstream regulators is lacking. Data from a well-characterized tumor tissue microarray were used for a Bayesian network analysis using the max-min hill-climbing method. In non-malignant tissue samples, a directionality of pEGFR (the phosphorylated form of the epidermal growth factor receptor) → CB1 receptors were found regardless as to whether the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) was included as a parameter. A similar result was found in the tumor tissue, but only when FAAH was included in the analysis. A second regulatory pathway, from the growth factor receptor ErbB2 → FAAH was also identified in the tumor samples. Transfection of AT1 prostate cancer cells with CB1 receptors induced a sensitivity to the growth-inhibiting effects of the CB receptor agonist CP55,940. The sensitivity was not dependent upon the level of receptor expression. Thus a high CB1 receptor expression alone does not drive the cells towards a survival phenotype in the presence of a CB receptor agonist. The data identify two potential regulators of the endocannabinoid system in prostate cancer and allow the construction of a model of a dysregulated endocannabinoid signaling network in this tumor. Further studies should be designed to test the veracity of the predictions of the network analysis in prostate cancer and other solid tumors. © 2014 The Authors. The Prostate published by Wiley Periodicals, Inc.

Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

PubMed

Sayar, Nilufer; Karahan, Gurbet; Konu, Ozlen; Bozkurt, Betul; Bozdogan, Onder; Yulug, Isik G

2015-01-01

CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

Circular RNA In Invasive and Recurrent Clinical Nonfunctioning Pituitary Adenomas: Expression Profiles and Bioinformatic Analysis.

PubMed

Wang, Jianpeng; Wang, Dong; Wan, Dehong; Ma, Qingxia; Liu, Qian; Li, Jiye; Li, Zhaojian; Gao, Yang; Jiang, Guohui; Ma, Leina; Liu, Jia; Li, Chuzhong

2018-06-14

The invasion and recurrence of clinical nonfunctioning pituitary adenomas (NFA) often lead to surgical treatment failure. Circular RNAs (circRNAs) are a novel class of RNAs whose 3' and 5' ends are joined together and have been shown to play important roles in cancer development. Up to now, the roles of circRNAs remain unclear in invasive and recurrent NFA. We detected and summarized the circRNA expression pattern in 75 NFA tissues from 10 non-invasive cases and 65 invasive cases and 9 pairs NFA tumor tissues from 9 recurrent cases by circRNA microarrays. Accordingly, functional enrichment analysis and pathway analysis were performed and circRNA-microRNA(miRNA) network were generated by bioinformatic analysis tools. 5 new invasive NFA samples and 5 non-invasive NFA samples were collected to measure the microarray results. 570 dysregulated circRNAs (Invasive Tumor vs. Non-invasive Tumor) and 10 up-regulated circRNAs (Recurrent tumor Tissue vs. First surgery tumor Tissue) were identified based on the situation (FC>2, P<0.05). The parental genes of the dysregulated circRNAs in the comparison between invasion tumor and non-invasion tumor were found to be enriched in some cell adhesion signaling pathways such as Focal adhesion, Hippo signaling pathway, PI3K-Akt signaling pathway, and Adherens junction. The circRNA-miRNA network showed that the dysregulated circRNA may function as miRNA sponges. This is the first study to conduct and comprehensively analyze the circRNA expression profile in invasive and recurrent NFA. Our finding will provide evidence for the significance of circRNAs in NFA diagnosis, prognosis and clinical treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Upregulation of the ESR1 Gene and ESR Ratio (ESR1/ESR2) is Associated with a Worse Prognosis in Papillary Thyroid Carcinoma: The Impact of the Estrogen Receptor α/β Expression on Clinical Outcomes in Papillary Thyroid Carcinoma Patients.

PubMed

Yi, Jin Wook; Kim, Su-Jin; Kim, Jong Kyu; Seong, Chan Yong; Yu, Hyeong Won; Chai, Young Jun; Choi, June Young; Lee, Kyu Eun

2017-11-01

A gender disparity exists with respect to the incidence of papillary thyroid cancer (PTC), suggesting that sex hormones such as estrogen play a role in PTC development and progression. In this study, we compared estrogen receptor gene expression patterns in PTCs to determine the clinical significance of estrogen gene expression in PTC. We analyzed ESR1 and ESR2 messenger RNA expression counts using data from The Cancer Genome Atlas (TCGA). To validate the results of TCGA analysis, we analyzed microarray data (GSE 54958) from the Gene Expression Omnibus. ESR1 gene expression and ESR ratio (ESR1/ESR2) were significantly higher in PTC tissues than in paired normal thyroid tissues (mean 659.427 vs. 264.045 for ESR1, 92.017 vs. 19.064 for ESR ratio). Among female patients, ESR1 expression and ESR ratio were negatively correlated with increased age. ESR1 expression and ESR ratio were higher in patients with classic PTC, lymphovascular invasion, BRAF V600E mutation, and radioiodine therapy. Classification analysis demonstrated that higher ESR1 expression and a higher ESR ratio faced a worse overall survival (hazard ratio 6.348 for ESR1, 4.031 for ESR ratio). Validation microarray analysis demonstrated that ESR1 expression and ESR ratio were higher in tumor tissues, classic PTC, and BRAF V600E . Higher ESR1 expression and a higher ESR ratio were associated with aggressive prognostic factors and worse overall survival in female PTC patients. Our results suggest that ESR1 and ESR ratio can be used as prognostic markers to predict female patient survival and have potential as a therapeutic target.

Double immunohistochemical staining with MUC4/p53 is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray-based study.

PubMed

Bhardwaj, Atul; Marsh, William L; Nash, Jason W; Barbacioru, Catalin C; Jones, Susie; Frankel, Wendy L

2007-04-01

Immunohistochemical stains have been used for the distinction of pancreatic adenocarcinoma from chronic pancreatitis. To determine if a double stain for MUC/p53 improved specificity and sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis by comparing maspin, mucin 4 (MUC4), p53, Smad4, and the double stain MUC4/p53. Seventy-four pancreatic adenocarcinomas and 19 chronic pancreatitis cases were retrieved from archival files. Tissue cores were arrayed to create a tissue microarray of 2-mm cores. Sections were stained with antibodies against maspin, MUC4, p53, and Smad4. Additionally, a 2-color, double stain for MUC4 and p53 was developed and evaluated. Five percent or greater staining in either of the cores was considered positive. Intensity (0, 1, 2) and extent (%) of tumor cells staining was also determined. The sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis with maspin, MUC4, p53, and Smad4 was 90%, 77%, 60%, and 63%, respectively; the specificity was 67%, 78%, 88%, and 88%, respectively. When MUC4 and p53 were combined in a double stain, and positive staining for either considered a positive result, the sensitivity increased to 96% but specificity was 73%. When immunoreactivity for both antibodies was necessary for a positive result, sensitivity fell to 39% but specificity was 100%. No correlation was found between intensity or extent of staining with any of the individual stains and tumor differentiation. The double immunohistochemical stain for MUC4/p53 can be a useful diagnostic tool in conjunction with the hematoxylin-eosin-stained section for pancreatic adenocarcinoma, particularly when limited tumor is available for multiple stains.

Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays

PubMed Central

Sugnet, Charles W; Srinivasan, Karpagam; Clark, Tyson A; O'Brien, Georgeann; Cline, Melissa S; Wang, Hui; Williams, Alan; Kulp, David; Blume, John E; Haussler, David; Ares, Manuel

2006-01-01

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. PMID:16424921

Immunohistochemical localization of steroid receptor coactivators in chondrosarcoma: an in vivo tissue microarray study.

PubMed

Li, Wei; Fu, Jingshu; Bian, Chen; Zhang, Jiqiang; Xie, Zhao

2014-12-01

Chondrosarcoma is the second most common type of primary bone malignancy following up osteosarcoma, characterized by resistance to conventional chemotherapeutic agents and radiation regimens. The p160 family members steroid receptor coactivator-1 and -3 (SRC-1 and SRC-3) have been implied in the regulation of cancer growth, migration, invasion, metastasis and chemotherapeutic resistance; but we still lack detailed information about the levels of SRCs in chondrosarcoma. In this study, expression of SRC-1 and SRC-3 in chondrosarcoma was examined by immunohistochemistry with tissue microarrays; the four score system (0, 1, 2 and 3) was used to evaluate the staining. The results showed that there were no gender-, site- or age-differences regarding the expression of SRC-1 or SRC-3 (p>0.05); organ (bone or cartilage) -differences were only detected for SRC-1 but not SRC-3 (p<0.05). Significant higher levels of SRC-1 and SRC-3 were detected in MDC and PDC when compared to WDC. Our study clearly demonstrated differentiation-dependant expression of SRC-1 and SRC-3 in chondrosarcoma, may be novel targets for the prognosis and/or treatment of chondrosarcoma, would have opened a new avenue and established foundation for studying chondrosarcoma. Copyright © 2014 Elsevier GmbH. All rights reserved.

Comparison of hepatocellular carcinoma in American and Asian patients by tissue array analysis.

PubMed

Song, Tae-Jin; Fong, Yuman; Cho, Sung-Jin; Gönen, Mithat; Hezel, Michael; Tuorto, Scott; Choi, Sang-Yong; Kim, Young-Chul; Suh, Sung-Ock; Koo, Bum-Hwan; Chae, Yang-Seok; Jarnagin, William R; Klimstra, David S

2012-07-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although some epidemiologic and etiologic differences between Asian and Western HCC are known, detailed comparative studies with pathologic correlations have not been performed. Paraffin sections of resected HCC specimens from Memorial Sloan-Kettering Cancer Center and Korea University Medical Center were used to construct tissue microarrays. Immunohistochemical staining of microarray sections was performed using antibodies against markers of proliferation and regulators of cell cycle. Patient data were correlated with staining results. When comparing both cohorts, significant differences were found in expression of p53 and MDM2. In the Asian group, more frequent positive staining for p53 (24%) was observed compared with the American group (9%; P = 0.037). For MDM2, 26% of American cases stained positive compared with 2% of Asian cases (P = 0.0003). No significant differences were found in expression of Ki67, p21, p27, cyclin D1, or bcl2. Female gender, vascular invasion, and lack of viral hepatitis infection correlated with positive MDM2 staining. These data likely correlate with differences in molecular pathogenesis of HCC based on racial and regional differences. These findings may have implications in choice of molecular targeted therapies based on patient ethnicity. Copyright © 2012 Wiley Periodicals, Inc.

[Transciptome among Mexicans: a large scale methodology to analyze the genetics expression profile of simultaneous samples in muscle, adipose tissue and lymphocytes obtained from the same individual].

PubMed

Bastarrachea, Raúl A; López-Alvarenga, Juan Carlos; Kent, Jack W; Laviada-Molina, Hugo A; Cerda-Flores, Ricardo M; Calderón-Garcidueñas, Ana Laura; Torres-Salazar, Amada; Torres-Salazar, Amanda; Nava-González, Edna J; Solis-Pérez, Elizabeth; Gallegos-Cabrales, Esther C; Cole, Shelley A; Comuzzie, Anthony G

2008-01-01

We describe the methodology used to analyze multiple transcripts using microarray techniques in simultaneous biopsies of muscle, adipose tissue and lymphocytes obtained from the same individual as part of the standard protocol of the Genetics of Metabolic Diseases in Mexico: GEMM Family Study. We recruited 4 healthy male subjects with BM1 20-41, who signed an informed consent letter. Subjects participated in a clinical examination that included anthropometric and body composition measurements, muscle biopsies (vastus lateralis) subcutaneous fat biopsies anda blood draw. All samples provided sufficient amplified RNA for microarray analysis. Total RNA was extracted from the biopsy samples and amplified for analysis. Of the 48,687 transcript targets queried, 39.4% were detectable in a least one of the studied tissues. Leptin was not detectable in lymphocytes, weakly expressed in muscle, but overexpressed and highly correlated with BMI in subcutaneous fat. Another example was GLUT4, which was detectable only in muscle and not correlated with BMI. Expression level concordance was 0.7 (p< 0.001) for the three tissues studied. We demonstrated the feasibility of carrying out simultaneous analysis of gene expression in multiple tissues, concordance of genetic expression in different tissues, and obtained confidence that this method corroborates the expected biological relationships among LEPand GLUT4. TheGEMM study will provide a broad and valuable overview on metabolic diseases, including obesity and type 2 diabetes.

Loss of Nuclear Localized and Tyrosine Phosphorylated Stat5 in Breast Cancer Predicts Poor Clinical Outcome and Increased Risk of Antiestrogen Therapy Failure

PubMed Central

Peck, Amy R.; Witkiewicz, Agnieszka K.; Liu, Chengbao; Stringer, Ginger A.; Klimowicz, Alexander C.; Pequignot, Edward; Freydin, Boris; Tran, Thai H.; Yang, Ning; Rosenberg, Anne L.; Hooke, Jeffrey A.; Kovatich, Albert J.; Nevalainen, Marja T.; Shriver, Craig D.; Hyslop, Terry; Sauter, Guido; Rimm, David L.; Magliocco, Anthony M.; Rui, Hallgeir

2011-01-01

Purpose To investigate nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) as a marker of prognosis in node-negative breast cancer and as a predictor of response to antiestrogen therapy. Patients and Methods Levels of Nuc-pYStat5 were analyzed in five archival cohorts of breast cancer by traditional diaminobenzidine-chromogen immunostaining and pathologist scoring of whole tissue sections or by immunofluorescence and automated quantitative analysis (AQUA) of tissue microarrays. Results Nuc-pYStat5 was an independent prognostic marker as measured by cancer-specific survival (CSS) in patients with node-negative breast cancer who did not receive systemic adjuvant therapy, when adjusted for common pathology parameters in multivariate analyses both by standard chromogen detection with pathologist scoring of whole tissue sections (cohort I; n = 233) and quantitative immunofluorescence of a tissue microarray (cohort II; n = 291). Two distinct monoclonal antibodies gave concordant results. A progression array (cohort III; n = 180) revealed frequent loss of Nuc-pYStat5 in invasive carcinoma compared to normal breast epithelia or ductal carcinoma in situ, and general loss of Nuc-pYStat5 in lymph node metastases. In cohort IV (n = 221), loss of Nuc-pYStat5 was associated with increased risk of antiestrogen therapy failure as measured by univariate CSS and time to recurrence (TTR). More sensitive AQUA quantification of Nuc-pYStat5 in antiestrogen-treated patients (cohort V; n = 97) identified by multivariate analysis patients with low Nuc-pYStat5 at elevated risk for therapy failure (CSS hazard ratio [HR], 21.55; 95% CI, 5.61 to 82.77; P < .001; TTR HR, 7.30; 95% CI, 2.34 to 22.78; P = .001). Conclusion Nuc-pYStat5 is an independent prognostic marker in node-negative breast cancer. If confirmed in prospective studies, Nuc-pYStat5 may become a useful predictive marker of response to adjuvant hormone therapy. PMID:21576635

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6

PubMed Central

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-01-01

AIM: To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. METHODS: Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. RESULTS: Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. CONCLUSION: MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection. PMID:27559432

Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice.

PubMed

Wang, Rui; Zhu, Menglin; Ye, Rongjian; Liu, Zuoxiong; Zhou, Fei; Chen, Hao; Lin, Yongjun

2015-12-11

As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice.

FlyAtlas: database of gene expression in the tissues of Drosophila melanogaster

PubMed Central

Robinson, Scott W.; Herzyk, Pawel; Dow, Julian A. T.; Leader, David P.

2013-01-01

The FlyAtlas resource contains data on the expression of the genes of Drosophila melanogaster in different tissues (currently 25—17 adult and 8 larval) obtained by hybridization of messenger RNA to Affymetrix Drosophila Genome 2 microarrays. The microarray probe sets cover 13 250 Drosophila genes, detecting 12 533 in an unambiguous manner. The data underlying the original web application (http://flyatlas.org) have been restructured into a relational database and a Java servlet written to provide a new web interface, FlyAtlas 2 (http://flyatlas.gla.ac.uk/), which allows several additional queries. Users can retrieve data for individual genes or for groups of genes belonging to the same or related ontological categories. Assistance in selecting valid search terms is provided by an Ajax ‘autosuggest’ facility that polls the database as the user types. Searches can also focus on particular tissues, and data can be retrieved for the most highly expressed genes, for genes of a particular category with above-average expression or for genes with the greatest difference in expression between the larval and adult stages. A novel facility allows the database to be queried with a specific gene to find other genes with a similar pattern of expression across the different tissues. PMID:23203866

FlyAtlas: database of gene expression in the tissues of Drosophila melanogaster.

PubMed

Robinson, Scott W; Herzyk, Pawel; Dow, Julian A T; Leader, David P

2013-01-01

The FlyAtlas resource contains data on the expression of the genes of Drosophila melanogaster in different tissues (currently 25-17 adult and 8 larval) obtained by hybridization of messenger RNA to Affymetrix Drosophila Genome 2 microarrays. The microarray probe sets cover 13,250 Drosophila genes, detecting 12,533 in an unambiguous manner. The data underlying the original web application (http://flyatlas.org) have been restructured into a relational database and a Java servlet written to provide a new web interface, FlyAtlas 2 (http://flyatlas.gla.ac.uk/), which allows several additional queries. Users can retrieve data for individual genes or for groups of genes belonging to the same or related ontological categories. Assistance in selecting valid search terms is provided by an Ajax 'autosuggest' facility that polls the database as the user types. Searches can also focus on particular tissues, and data can be retrieved for the most highly expressed genes, for genes of a particular category with above-average expression or for genes with the greatest difference in expression between the larval and adult stages. A novel facility allows the database to be queried with a specific gene to find other genes with a similar pattern of expression across the different tissues.

Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer

PubMed Central

Gao, Yan; Foster, Rosemary; Yang, Xiaoqian; Feng, Yong; Shen, Jacson K.; Mankin, Henry J.; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

2015-01-01

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer. PMID:25823654

Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer.

PubMed

Gao, Yan; Foster, Rosemary; Yang, Xiaoqian; Feng, Yong; Shen, Jacson K; Mankin, Henry J; Hornicek, Francis J; Amiji, Mansoor M; Duan, Zhenfeng

2015-04-20

The clinical significance of Cluster of Differentiation 44 (CD44) remains controversial in human ovarian cancer. The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer. In this study, a unique ovarian cancer tissue microarray (TMA) was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. CD44 expression in TMA was assessed by immunohistochemistry. Both the metastatic and recurrent ovarian cancer tissues expressed higher level of CD44 than the patient-matched primary tumor. A significant association has been shown between CD44 expression and both the disease free survival and overall survival. A strong increase of CD44 was found in the tumor recurrence of mouse model. Finally, when CD44 was knocked down, proliferation, migration/invasion activity, and spheroid formation were significantly suppressed, while drug sensitivity was enhanced. Thus, up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. Developing strategies to target CD44 may prevent metastasis, recurrence, and drug resistance in ovarian cancer.

Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

PubMed

Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W

2015-11-01

Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combining Evidence of Preferential Gene-Tissue Relationships from Multiple Sources

PubMed Central

Guo, Jing; Hammar, Mårten; Öberg, Lisa; Padmanabhuni, Shanmukha S.; Bjäreland, Marcus; Dalevi, Daniel

2013-01-01

An important challenge in drug discovery and disease prognosis is to predict genes that are preferentially expressed in one or a few tissues, i.e. showing a considerably higher expression in one tissue(s) compared to the others. Although several data sources and methods have been published explicitly for this purpose, they often disagree and it is not evident how to retrieve these genes and how to distinguish true biological findings from those that are due to choice-of-method and/or experimental settings. In this work we have developed a computational approach that combines results from multiple methods and datasets with the aim to eliminate method/study-specific biases and to improve the predictability of preferentially expressed human genes. A rule-based score is used to merge and assign support to the results. Five sets of genes with known tissue specificity were used for parameter pruning and cross-validation. In total we identify 3434 tissue-specific genes. We compare the genes of highest scores with the public databases: PaGenBase (microarray), TiGER (EST) and HPA (protein expression data). The results have 85% overlap to PaGenBase, 71% to TiGER and only 28% to HPA. 99% of our predictions have support from at least one of these databases. Our approach also performs better than any of the databases on identifying drug targets and biomarkers with known tissue-specificity. PMID:23950964

Circular RNA profiles in mouse lung tissue induced by radon.

PubMed

Pei, Weiwei; Tao, Lijing; Zhang, Leshuai W; Zhang, Shuyu; Cao, Jianping; Jiao, Yang; Tong, Jian; Nie, Jihua

2017-04-07

Radon is a known human lung carcinogen, whose underlying carcinogenic mechanism remains unclear. Recently, circular RNA (circRNA), a class of endogenous non-protein coding RNAs that contain a circular loop, was found to exhibit multiple biological effects. In this study, circRNA profiles in mouse lung tissues between control and radon exposure were analyzed. Six mice were exposed to radon at concentration of 100,000 Bq/m 3 , 12 h/d, for up to cumulative doses of 60 working level months (WLM). H&E staining and immunohistochemistry of caspase-3 were used to detect the damages in lung tissue. The lung tissue of control and exposed group were selected for circRNA microarray study. The circRNA/microRNA interaction was analyzed by starBase prediction software. 5 highest expressing circRNAs were selected by real-time PCR to validate the consistency in mouse lung tissue exposed to radon. Inflammatory reaction was found in mouse lung tissue exposed to radon, and caspase-3 expression was significantly increased. Microarray screening revealed 107 up-regulated and 83 down-regulated circRNAs, among which top 30 circRNAs with the highest fold changes were chosen for further analysis, with 5 microRNAs binding sites listed for each circRNA. Consistency of the top 5 circRNAs with the highest expressions were confirmed in mice exposed with 60WLM of radon. Mouse lung tissue was severely injured when exposed to radon through pathological diagnosis and immunohistochemical analysis. A series of differentially expressed circRNAs demonstrated that they may play an important role in pulmonary toxicity induced by radon.

Implementation of spectral clustering with partitioning around medoids (PAM) algorithm on microarray data of carcinoma

NASA Astrophysics Data System (ADS)

Cahyaningrum, Rosalia D.; Bustamam, Alhadi; Siswantining, Titin

2017-03-01

Technology of microarray became one of the imperative tools in life science to observe the gene expression levels, one of which is the expression of the genes of people with carcinoma. Carcinoma is a cancer that forms in the epithelial tissue. These data can be analyzed such as the identification expressions hereditary gene and also build classifications that can be used to improve diagnosis of carcinoma. Microarray data usually served in large dimension that most methods require large computing time to do the grouping. Therefore, this study uses spectral clustering method which allows to work with any object for reduces dimension. Spectral clustering method is a method based on spectral decomposition of the matrix which is represented in the form of a graph. After the data dimensions are reduced, then the data are partitioned. One of the famous partition method is Partitioning Around Medoids (PAM) which is minimize the objective function with exchanges all the non-medoid points into medoid point iteratively until converge. Objectivity of this research is to implement methods spectral clustering and partitioning algorithm PAM to obtain groups of 7457 genes with carcinoma based on the similarity value. The result in this study is two groups of genes with carcinoma.

Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens.

PubMed

Liebe, Sebastian; Christ, Daniela S; Ehricht, Ralf; Varrelmann, Mark

2016-01-01

Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present.

A gene expression signature associated with survival in metastatic melanoma

PubMed Central

Mandruzzato, Susanna; Callegaro, Andrea; Turcatel, Gianluca; Francescato, Samuela; Montesco, Maria C; Chiarion-Sileni, Vanna; Mocellin, Simone; Rossi, Carlo R; Bicciato, Silvio; Wang, Ena; Marincola, Francesco M; Zanovello, Paola

2006-01-01

Background Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival. Methods Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM) to identify genes associated with patient survival, and supervised principal components (SPC) to determine survival prediction. Results SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival. Conclusion The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells. PMID:17129373

Response of sweet orange (Citrus sinensis) to 'Candidatus Liberibacter asiaticus' infection: microscopy and microarray analyses.

PubMed

Kim, Jeong-Soon; Sagaram, Uma Shankar; Burns, Jacqueline K; Li, Jian-Liang; Wang, Nian

2009-01-01

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium 'Candidatus Liberibacter spp.' In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since 'Ca. Liberibacter asiaticus' does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

Thermodynamically optimal whole-genome tiling microarray design and validation.

PubMed

Cho, Hyejin; Chou, Hui-Hsien

2016-06-13

Microarray is an efficient apparatus to interrogate the whole transcriptome of species. Microarray can be designed according to annotated gene sets, but the resulted microarrays cannot be used to identify novel transcripts and this design method is not applicable to unannotated species. Alternatively, a whole-genome tiling microarray can be designed using only genomic sequences without gene annotations, and it can be used to detect novel RNA transcripts as well as known genes. The difficulty with tiling microarray design lies in the tradeoff between probe-specificity and coverage of the genome. Sequence comparison methods based on BLAST or similar software are commonly employed in microarray design, but they cannot precisely determine the subtle thermodynamic competition between probe targets and partially matched probe nontargets during hybridizations. Using the whole-genome thermodynamic analysis software PICKY to design tiling microarrays, we can achieve maximum whole-genome coverage allowable under the thermodynamic constraints of each target genome. The resulted tiling microarrays are thermodynamically optimal in the sense that all selected probes share the same melting temperature separation range between their targets and closest nontargets, and no additional probes can be added without violating the specificity of the microarray to the target genome. This new design method was used to create two whole-genome tiling microarrays for Escherichia coli MG1655 and Agrobacterium tumefaciens C58 and the experiment results validated the design.

Importing MAGE-ML format microarray data into BioConductor.

PubMed

Durinck, Steffen; Allemeersch, Joke; Carey, Vincent J; Moreau, Yves; De Moor, Bart

2004-12-12

The microarray gene expression markup language (MAGE-ML) is a widely used XML (eXtensible Markup Language) standard for describing and exchanging information about microarray experiments. It can describe microarray designs, microarray experiment designs, gene expression data and data analysis results. We describe RMAGEML, a new Bioconductor package that provides a link between cDNA microarray data stored in MAGE-ML format and the Bioconductor framework for preprocessing, visualization and analysis of microarray experiments. http://www.bioconductor.org. Open Source.

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

PubMed

Haas, Christian S; Creighton, Chad J; Pi, Xiujun; Maine, Ira; Koch, Alisa E; Haines, G Kenneth; Ling, Song; Chinnaiyan, Arul M; Holoshitz, Joseph

2006-07-01

To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

Diffuse myogenin expression by immunohistochemistry is an independent marker of poor survival in pediatric rhabdomyosarcoma: a tissue microarray study of 71 primary tumors including correlation with molecular phenotype.

PubMed

Heerema-McKenney, Amy; Wijnaendts, Liliane C D; Pulliam, Joseph F; Lopez-Terrada, Dolores; McKenney, Jesse K; Zhu, Shirley; Montgomery, Kelli; Mitchell, Janet; Marinelli, Robert J; Hart, Augustinus A M; van de Rijn, Matt; Linn, Sabine C

2008-10-01

The pathologic classification of rhabdomyosarcoma (RMS) into embryonal or alveolar subtype is an important prognostic factor guiding the therapeutic protocol chosen for an individual patient. Unfortunately, this classification is not always straightforward, and the diagnostic criteria are controversial in a subset of cases. Ancillary studies are used to aid in the classification, but their potential use as independent prognostic factors is rarely studied. The aim of this study is to identify immunohistochemical markers of potential prognostic significance in pediatric RMS and to correlate their expression with PAX-3/FKHR and PAX-7/FKHR fusion status. A single tissue microarray containing 71 paraffin-embedded pediatric RMSs was immunostained with antibodies against p53, bcl-2, Ki-67, CD44, myogenin, and MyoD1. The tissue microarray and whole paraffin blocks were studied for PAX-3/FKHR and PAX-7/FKHR gene fusions by fluorescence in situ hybridization and reverse transcription-polymerase chain reaction. Clinical follow-up data were available for each patient. Immunohistochemical staining results and translocation status were correlated with recurrence-free interval (RFI) and overall survival (OS) using the Kaplan-Meier method, the log-rank test, and Cox proportional hazard regression. The minimum clinical follow-up interval was 24 months (median follow-up=57 mo). On univariable analysis, immunohistochemical expression of myogenin, bcl-2, and identification of a gene fusion were associated with decreased 5-year RFI and 10-year OS (myogenin RFI P=0.0028, OS P=0.0021; bcl-2 RFI P=0.037, OS P=0.032; gene fusion RFI P=0.0001, OS P=0.0058). After adjustment for Intergroup Rhabdomyosarcoma Study-TNM stage, tumor site, age, tumor histology, and translocation status by multivariable analysis, only myogenin retained an independent association with RFI (P=0.034) and OS (P=0.0069). In this retrospective analysis, diffuse immunohistochemical reactivity for myogenin in RMS correlates with decreased RFI and OS, independent of histologic subtype, translocation status, tumor site, or stage.

Cytokine-related genes and oxidation-related genes detected in preeclamptic placentas.

PubMed

Lee, Gui Se Ra; Joe, Yoon Seong; Kim, Sa Jin; Shin, Jong Chul

2010-10-01

To investigate cytokine- and oxidation-related genes for preeclampsia using DNA microarray analysis. Placentas were collected from 13 normal pregnancies and 13 patients with preeclampsia. Gene expression was studied using DNA microarray. Among significantly expressed genes, we focused on genes associated with cytokines and oxidation, and the results were confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). 415 genes out of 30,940 genes were altered by > or =2-fold in the microarray analysis. 121 up-regulated genes and 294 down-regulated genes were found to be in preeclamptic placenta. Six cytokine-related genes and 5 oxidation-related genes were found from among the 121 up-regulated genes. The cytokine-related genes studied included oncostatin M (OSM), fms-related tyrosine kinase (FLT1) and vascular endothelial growth factor A (VEGFA), and the oxidation-related genes studied included spermine oxidase (SMOX), l cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1), acetate dehydrogenase A (LDHA). These six genes were also significantly higher in placentas from patients with preeclampsia than in those from women with normal pregnancies. The placental tissue of patients with preeclampsia showed significantly higher mRNA expression of these six genes than the normal group, using QRT-PCR. DNA microarray analysis is one of the great methods for simultaneously detecting the functionally associated genes of preeclampsia. The cytokine-related genes such as OSM, FLT1 and VEGFA, and the oxidation-related genes such as LDHA, CYP26A1 and SMOX might prove to be the starting point in the elucidation of the pathogenesis of preeclampsia.

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

PubMed

De-Ugarte, Laura; Serra-Vinardell, Jenny; Nonell, Lara; Balcells, Susana; Arnal, Magdalena; Nogues, Xavier; Mellibovsky, Leonardo; Grinberg, Daniel; Diez-Perez, Adolfo; Garcia-Giralt, Natalia

2018-01-01

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

A mixture model-based approach to the clustering of microarray expression data.

PubMed

McLachlan, G J; Bean, R W; Peel, D

2002-03-01

This paper introduces the software EMMIX-GENE that has been developed for the specific purpose of a model-based approach to the clustering of microarray expression data, in particular, of tissue samples on a very large number of genes. The latter is a nonstandard problem in parametric cluster analysis because the dimension of the feature space (the number of genes) is typically much greater than the number of tissues. A feasible approach is provided by first selecting a subset of the genes relevant for the clustering of the tissue samples by fitting mixtures of t distributions to rank the genes in order of increasing size of the likelihood ratio statistic for the test of one versus two components in the mixture model. The imposition of a threshold on the likelihood ratio statistic used in conjunction with a threshold on the size of a cluster allows the selection of a relevant set of genes. However, even this reduced set of genes will usually be too large for a normal mixture model to be fitted directly to the tissues, and so the use of mixtures of factor analyzers is exploited to reduce effectively the dimension of the feature space of genes. The usefulness of the EMMIX-GENE approach for the clustering of tissue samples is demonstrated on two well-known data sets on colon and leukaemia tissues. For both data sets, relevant subsets of the genes are able to be selected that reveal interesting clusterings of the tissues that are either consistent with the external classification of the tissues or with background and biological knowledge of these sets. EMMIX-GENE is available at http://www.maths.uq.edu.au/~gjm/emmix-gene/

ModuleMiner - improved computational detection of cis-regulatory modules: are there different modes of gene regulation in embryonic development and adult tissues?

PubMed Central

Van Loo, Peter; Aerts, Stein; Thienpont, Bernard; De Moor, Bart; Moreau, Yves; Marynen, Peter

2008-01-01

We present ModuleMiner, a novel algorithm for computationally detecting cis-regulatory modules (CRMs) in a set of co-expressed genes. ModuleMiner outperforms other methods for CRM detection on benchmark data, and successfully detects CRMs in tissue-specific microarray clusters and in embryonic development gene sets. Interestingly, CRM predictions for differentiated tissues exhibit strong enrichment close to the transcription start site, whereas CRM predictions for embryonic development gene sets are depleted in this region. PMID:18394174

Transcriptome architecture across tissues in the pig

PubMed Central

Ferraz, André LJ; Ojeda, Ana; López-Béjar, Manel; Fernandes, Lana T; Castelló, Anna; Folch, Josep M; Pérez-Enciso, Miguel

2008-01-01

Background Artificial selection has resulted in animal breeds with extreme phenotypes. As an organism is made up of many different tissues and organs, each with its own genetic programme, it is pertinent to ask: How relevant is tissue in terms of total transcriptome variability? Which are the genes most distinctly expressed between tissues? Does breed or sex equally affect the transcriptome across tissues? Results In order to gain insight on these issues, we conducted microarray expression profiling of 16 different tissues from four animals of two extreme pig breeds, Large White and Iberian, two males and two females. Mixed model analysis and neighbor – joining trees showed that tissues with similar developmental origin clustered closer than those with different embryonic origins. Often a sound biological interpretation was possible for overrepresented gene ontology categories within differentially expressed genes between groups of tissues. For instance, an excess of nervous system or muscle development genes were found among tissues of ectoderm or mesoderm origins, respectively. Tissue accounted for ~11 times more variability than sex or breed. Nevertheless, we were able to confidently identify genes with differential expression across tissues between breeds (33 genes) and between sexes (19 genes). The genes primarily affected by sex were overall different than those affected by breed or tissue. Interaction with tissue can be important for differentially expressed genes between breeds but not so much for genes whose expression differ between sexes. Conclusion Embryonic development leaves an enduring footprint on the transcriptome. The interaction in gene × tissue for differentially expressed genes between breeds suggests that animal breeding has targeted differentially each tissue's transcriptome. PMID:18416811

Genomic DNA Hypomethylation Is Associated with Neural Tube Defects Induced by Methotrexate Inhibition of Folate Metabolism

PubMed Central

Wang, Xiuwei; Guan, Zhen; Chen, Yan; Dong, Yanting; Niu, Yuhu; Wang, Jianhua; Zhang, Ting; Niu, Bo

2015-01-01

DNA methylation is thought to be involved in the etiology of neural tube defects (NTDs). However, the exact mechanism between DNA methylation and NTDs remains unclear. Herein, we investigated the change of methylation in mouse model of NTDs associated with folate dysmetabolism by use of ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS), liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS), microarray, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Real time quantitative PCR. Results showed that NTD neural tube tissues had lower concentrations of 5-methyltetrahydrofolate (5-MeTHF, P = 0.005), 5-formyltetrahydrofolate (5-FoTHF, P = 0.040), S-adenosylmethionine (SAM, P = 0.004) and higher concentrations of folic acid (P = 0.041), homocysteine (Hcy, P = 0.006) and S-adenosylhomocysteine (SAH, P = 0.045) compared to control. Methylation levels of genomic DNA decreased significantly in the embryonic neural tube tissue of NTD samples. 132 differentially methylated regions (35 low methylated regions and 97 high methylated regions) were selected by microarray. Two genes (Siah1b, Prkx) in Wnt signal pathway demonstrated lower methylated regions (peak) and higher expression in NTDs (P<0.05; P<0.05). Results suggest that DNA hypomethylation was one of the possible epigenetic variations correlated with the occurrence of NTDs induced by folate dysmetabolism and that Siah1b, Prkx in Wnt pathway may be candidate genes for NTDs. PMID:25822193

Robust Segmentation of Overlapping Cells in Histopathology Specimens Using Parallel Seed Detection and Repulsive Level Set

PubMed Central

Qi, Xin; Xing, Fuyong; Foran, David J.; Yang, Lin

2013-01-01

Automated image analysis of histopathology specimens could potentially provide support for early detection and improved characterization of breast cancer. Automated segmentation of the cells comprising imaged tissue microarrays (TMA) is a prerequisite for any subsequent quantitative analysis. Unfortunately, crowding and overlapping of cells present significant challenges for most traditional segmentation algorithms. In this paper, we propose a novel algorithm which can reliably separate touching cells in hematoxylin stained breast TMA specimens which have been acquired using a standard RGB camera. The algorithm is composed of two steps. It begins with a fast, reliable object center localization approach which utilizes single-path voting followed by mean-shift clustering. Next, the contour of each cell is obtained using a level set algorithm based on an interactive model. We compared the experimental results with those reported in the most current literature. Finally, performance was evaluated by comparing the pixel-wise accuracy provided by human experts with that produced by the new automated segmentation algorithm. The method was systematically tested on 234 image patches exhibiting dense overlap and containing more than 2200 cells. It was also tested on whole slide images including blood smears and tissue microarrays containing thousands of cells. Since the voting step of the seed detection algorithm is well suited for parallelization, a parallel version of the algorithm was implemented using graphic processing units (GPU) which resulted in significant speed-up over the C/C++ implementation. PMID:22167559

Activity of the Monocarboxylate Transporter 1 inhibitor AZD3965 in Small Cell Lung Cancer

PubMed Central

Polański, Radosław; Hodgkinson, Cassandra L.; Fusi, Alberto; Nonaka, Daisuke; Priest, Lynsey; Kelly, Paul; Trapani, Francesca; Bishop, Paul W.; White, Anne; Critchlow, Susan E.; Smith, Paul D.; Blackhall, Fiona

2013-01-01

Purpose The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 is undergoing Phase I evaluation in the UK. AZD3965 is proposed, via lactate transport modulation, to kill tumor cells reliant on glycolysis. We investigated the therapeutic potential of AZD3965 in small cell lung cancer (SCLC) seeking rationale for clinical testing in this disease and putative predictive biomarkers for trial use. Experimental Design AZD3965 sensitivity was determined for 7 SCLC cell lines, in normoxia and hypoxia, and for a tumor xenograft model. Proof of mechanism was sought via changes in intracellular/tumor lactate. Expression of MCT1 and related transporter MCT4 were assessed by western blot. Drug resistance was investigated via MCT4 siRNAi and overexpression. The expression and clinical significance of MCT1 and MCT4 were explored in a tissue microarray from 78 SCLC patients. Results AZD3965 sensitivity varied in vitro and was highest in hypoxia. Resistance in hypoxia was associated with increased MCT4 expression. In vivo, AZD3965 reduced tumor growth and increased intra-tumor lactate. In the tissue microarray, high MCT1 expression was associated with worse prognosis (p=0.014). MCT1 and hypoxia marker CA IX expression in the absence of MCT4 was observed in 21% of SCLC tumors. Conclusions This study provides a rationale to test AZD3965 in SCLC patients. Our results suggest that patients with tumors expressing MCT1 and lacking in MCT4 are most likely to respond. PMID:24277449

Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis

PubMed Central

Tsujikawa, Takahiro; Kumar, Sushil; Borkar, Rohan N.; Azimi, Vahid; Thibault, Guillaume; Chang, Young Hwan; Balter, Ariel; Kawashima, Rie; Choe, Gina; Sauer, David; El Rassi, Edward; Clayburgh, Daniel R.; Kulesz-Martin, Molly F.; Lutz, Eric R.; Zheng, Lei; Jaffee, Elizabeth M.; Leyshock, Patrick; Margolin, Adam A.; Mori, Motomi; Gray, Joe W.; Flint, Paul W.; Coussens, Lisa M.

2017-01-01

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment. PMID:28380359

Using pathway modules as targets for assay development in xenobiotic screening

EPA Science Inventory

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...

Molecular and physiological responses to titanium dioxide and cerium oxide nanoparticles in Arabidopsis

EPA Science Inventory

- Changes in tissue transcriptomes and productivity of Arabidopsis thaliana were investigated during exposure of plants to two widely-used engineered metal oxide nanoparticles, titanium dioxide (nano-titanium) and cerium dioxide (nano-cerium). Microarray analyses confirmed that e...

Met Nuclear Localization and Signaling in Breast Cancer

DTIC Science & Technology

2006-05-01

and in germinal regions of many tissues using 4 unique antibodies . Cell fractionation reveals a 60kDa band recognized by C-terminal Met antibodies ...cascades such as Gab1 , Grb2 and PI3K, leading to proliferation, scattering, increased motility, invasion and branching morphogenesis (reviewed in (2...Identification of Met antibodies for use on tissue microarray of normal and cancerous cells, Months 12-24 Task 2. Definition of the domain

Development and validation of a mixed-tissue oligonucleotide DNA microarray for Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758).

PubMed

Trumbić, Željka; Bekaert, Michaël; Taggart, John B; Bron, James E; Gharbi, Karim; Mladineo, Ivona

2015-11-25

The largest of the tuna species, Atlantic bluefin tuna (Thunnus thynnus), inhabits the North Atlantic Ocean and the Mediterranean Sea and is considered to be an endangered species, largely a consequence of overfishing. T. thynnus aquaculture, referred to as fattening or farming, is a capture based activity dependent on yearly renewal from the wild. Thus, the development of aquaculture practices independent of wild resources can provide an important contribution towards ensuring security and sustainability of this species in the longer-term. The development of such practices is today greatly assisted by large scale transcriptomic studies. We have used pyrosequencing technology to sequence a mixed-tissue normalised cDNA library, derived from adult T. thynnus. A total of 976,904 raw sequence reads were assembled into 33,105 unique transcripts having a mean length of 893 bases and an N50 of 870. Of these, 33.4% showed similarity to known proteins or gene transcripts and 86.6% of them were matched to the congeneric Pacific bluefin tuna (Thunnus orientalis) genome, compared to 70.3% for the more distantly related Nile tilapia (Oreochromis niloticus) genome. Transcript sequences were used to develop a novel 15 K Agilent oligonucleotide DNA microarray for T. thynnus and comparative tissue gene expression profiles were inferred for gill, heart, liver, ovaries and testes. Functional contrasts were strongest between gills and ovaries. Gills were particularly associated with immune system, signal transduction and cell communication, while ovaries displayed signatures of glycan biosynthesis, nucleotide metabolism, transcription, translation, replication and repair. Sequence data generated from a novel mixed-tissue T. thynnus cDNA library provide an important transcriptomic resource that can be further employed for study of various aspects of T. thynnus ecology and genomics, with strong applications in aquaculture. Tissue-specific gene expression profiles inferred through the use of novel oligo-microarray can serve in the design of new and more focused transcriptomic studies for future research of tuna physiology and assessment of the welfare in a production environment.

High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples.

PubMed

Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

2016-01-01

Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F.

Statistical issues in signal extraction from microarrays

NASA Astrophysics Data System (ADS)

Bergemann, Tracy; Quiaoit, Filemon; Delrow, Jeffrey J.; Zhao, Lue Ping

2001-06-01

Microarray technologies are increasingly used in biomedical research to study genome-wide expression profiles in the post genomic era. Their popularity is largely due to their high throughput and economical affordability. For example, microarrays have been applied to studies of cell cycle, regulatory circuitry, cancer cell lines, tumor tissues, and drug discoveries. One obstacle facing the continued success of applying microarray technologies, however, is the random variaton present on microarrays: within signal spots, between spots and among chips. In addition, signals extracted by available software packages seem to vary significantly. Despite a variety of software packages, it appears that there are two major approaches to signal extraction. One approach is to focus on the identification of signal regions and hence estimation of signal levels above background levels. The other approach is to use the distribution of intensity values as a way of identifying relevant signals. Building upon both approaches, the objective of our work is to develop a method that is statistically rigorous and also efficient and robust. Statistical issues to be considered here include: (1) how to refine grid alignment so that the overall variation is minimized, (2) how to estimate the signal levels relative to the local background levels as well as the variance of this estimate, and (3) how to integrate red and green channel signals so that the ratio of interest is stable, simultaneously relaxing distributional assumptions.

Population effect model identifies gene expression predictors of survival outcomes in lung adenocarcinoma for both Caucasian and Asian patients

PubMed Central

Cai, Guoshuai; Xiao, Feifei; Cheng, Chao; Li, Yafang; Amos, Christopher I.; Whitfield, Michael L.

2017-01-01

Background We analyzed and integrated transcriptome data from two large studies of lung adenocarcinomas on distinct populations. Our goal was to investigate the variable gene expression alterations between paired tumor-normal tissues and prospectively identify those alterations that can reliably predict lung disease related outcomes across populations. Methods We developed a mixed model that combined the paired tumor-normal RNA-seq from two populations. Alterations in gene expression common to both populations were detected and validated in two independent DNA microarray datasets. A 10-gene prognosis signature was developed through a l1 penalized regression approach and its prognostic value was evaluated in a third independent microarray cohort. Results Deregulation of apoptosis pathways and increased expression of cell cycle pathways were identified in tumors of both Caucasian and Asian lung adenocarcinoma patients. We demonstrate that a 10-gene biomarker panel can predict prognosis of lung adenocarcinoma in both Caucasians and Asians. Compared to low risk groups, high risk groups showed significantly shorter overall survival time (Caucasian patients data: HR = 3.63, p-value = 0.007; Asian patients data: HR = 3.25, p-value = 0.001). Conclusions This study uses a statistical framework to detect DEGs between paired tumor and normal tissues that considers variances among patients and ethnicities, which will aid in understanding the common genes and signalling pathways with the largest effect sizes in ethnically diverse cohorts. We propose multifunctional markers for distinguishing tumor from normal tissue and prognosis for both populations studied. PMID:28426704

A molecular analysis by gene expression profiling reveals Bik/NBK overexpression in sporadic breast tumor samples of Mexican females

PubMed Central

García, Normand; Salamanca, Fabio; Astudillo-de la Vega, Horacio; Curiel-Quesada, Everardo; Alvarado, Isabel; Peñaloza, Rosenda; Arenas, Diego

2005-01-01

Background Breast cancer is one of the most frequent causes of death in Mexican women over 35 years of age. At molecular level, changes in many genetic networks have been reported as associated with this neoplasia. To analyze these changes, we determined gene expression profiles of tumors from Mexican women with breast cancer at different stages and compared these with those of normal breast tissue samples. Methods 32P-radiolabeled cDNA was synthesized by reverse transcription of mRNA from fresh sporadic breast tumor biopsies, as well as normal breast tissue. cDNA probes were hybridized to microarrays and expression levels registered using a phosphorimager. Expression levels of some genes were validated by real time RT-PCR and immunohistochemical assays. Results We identified two subgroups of tumors according to their expression profiles, probably related with cancer progression. Ten genes, unexpressed in normal tissue, were turned on in some tumors. We found consistent high expression of Bik gene in 14/15 tumors with predominant cytoplasmic distribution. Conclusion Recently, the product of the Bik gene has been associated with tumoral reversion in different neoplasic cell lines, and was proposed as therapy to induce apoptosis in cancers, including breast tumors. Even though a relationship among genes, for example those from a particular pathway, can be observed through microarrays, this relationship might not be sufficient to assign a definitive role to Bik in development and progression of the neoplasia. The findings herein reported deserve further investigation. PMID:16060964

Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression.

PubMed

Zu, Xuyu; Ma, Jun; Liu, Hongxia; Liu, Feng; Tan, Chunyan; Yu, Lingling; Wang, Jue; Xie, Zhenhua; Cao, Deliang; Jiang, Yuyang

2011-03-10

Pokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms. Tissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene. Pokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P < 0.0001). ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter. Pokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy.

Toxicity of Doxorubicin on Pig Liver After Chemoembolization with Doxorubicin-loaded Microspheres: A Pilot DNA-microarrays and Histology Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verret, Valentin, E-mail: valentin.verret@archimmed.com; Namur, Julien; Ghegediban, Saieda Homayra

The potential mechanisms accounting for the hepatotoxicity of doxorubicin-loaded microspheres in chemoembolization were examined by combining histology and DNA-microarray techniques.The left hepatic arteries of two pigs were embolized with 1 mL of doxorubicin-loaded (25 mg; (DoxMS)) or non-loaded (BlandMS) microspheres. The histopathological effects of the embolization were analyzed at 1 week. RNAs extracted from both the embolized and control liver areas were hybridized onto Agilent porcine microarrays. Genes showing significantly different expression (p < 0.01; fold-change > 2) between two groups were classified by biological process. At 1 week after embolization, DoxMS caused arterial and parenchymal necrosis in 51 andmore » 38 % of embolized vessels, respectively. By contrast, BlandMS did not cause any tissue damage. Up-regulated genes following embolization with DoxMS (vs. BlandMS, n = 353) were mainly involved in cell death, apoptosis, and metabolism of doxorubicin. Down-regulated genes (n = 120) were mainly related to hepatic functions, including enzymes of lipid and carbohydrate metabolisms. Up-regulated genes included genes related to cell proliferation (growth factors and transcription factors), tissue remodeling (MMPs and several collagen types), inflammatory reaction (interleukins and chemokines), and angiogenesis (angiogenic factors and HIF1a pathway), all of which play an important role in liver healing and regeneration. DoxMS caused lesions to the liver, provoked cell death, and disturbed liver metabolism. An inflammatory repair process with cell proliferation, tissue remodeling, and angiogenesis was rapidly initiated during the first week after chemoembolization. This pilot study provides a comprehensive method to compare different types of DoxMS in healthy animals or tumor models.« less

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

PubMed Central

Sella, Sabrina; Adami, Valentina; Amati, Eliana; Bernardi, Martina; Chieregato, Katia; Gatto, Pamela; Menarin, Martina; Pozzato, Alessandro; Pozzato, Gianantonio; Astori, Giuseppe

2018-01-01

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling. PMID:29293552

Women's experiences receiving abnormal prenatal chromosomal microarray testing results.

PubMed

Bernhardt, Barbara A; Soucier, Danielle; Hanson, Karen; Savage, Melissa S; Jackson, Laird; Wapner, Ronald J

2013-02-01

Genomic microarrays can detect copy-number variants not detectable by conventional cytogenetics. This technology is diffusing rapidly into prenatal settings even though the clinical implications of many copy-number variants are currently unknown. We conducted a qualitative pilot study to explore the experiences of women receiving abnormal results from prenatal microarray testing performed in a research setting. Participants were a subset of women participating in a multicenter prospective study "Prenatal Cytogenetic Diagnosis by Array-based Copy Number Analysis." Telephone interviews were conducted with 23 women receiving abnormal prenatal microarray results. We found that five key elements dominated the experiences of women who had received abnormal prenatal microarray results: an offer too good to pass up, blindsided by the results, uncertainty and unquantifiable risks, need for support, and toxic knowledge. As prenatal microarray testing is increasingly used, uncertain findings will be common, resulting in greater need for careful pre- and posttest counseling, and more education of and resources for providers so they can adequately support the women who are undergoing testing.

Prediction of gene expression in embryonic structures of Drosophila melanogaster.

PubMed

Samsonova, Anastasia A; Niranjan, Mahesan; Russell, Steven; Brazma, Alvis

2007-07-01

Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms.

Prediction of Gene Expression in Embryonic Structures of Drosophila melanogaster

PubMed Central

Samsonova, Anastasia A; Niranjan, Mahesan; Russell, Steven; Brazma, Alvis

2007-01-01

Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms. PMID:17658945

Profile of differentially expressed miRNAs in high-grade serous carcinoma and clear cell ovarian carcinoma, and the expression of miR-510 in ovarian carcinoma

PubMed Central

ZHANG, XINCHEN; GUO, GORDON; WANG, GUANG; ZHAO, JINYAO; WANG, BO; YU, XIAOTANG; DING, YANFANG

2015-01-01

Improved insight into the molecular and genetic profile of different types of epithelial ovarian cancer (EOC) is required for understanding the carcinogenesis of EOC and may potentially be exploited by future targeted therapies. The aim of the present study was to identify a unique microRNA (miRNA) patterns and key miRNAs, which may assist in predicting progression and prognosis in high-grade serous carcinoma (HGSC) and clear cell carcinoma (CCC). To identify unique miRNA patterns associated with HGSC and CCC, a miRNA microarray was performed using Chinese tumor bank specimens of patients with HGSC or CCC in a retrospective analysis. The expression levels of four deregulated miRNAs were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in an external cohort of 42 cases of HGSC and 36 cases of CCC. Kaplan-Meier analysis was performed to analyze the correlation between the expression levels of the four miRNAs and patient prognosis. Among these validated miRNAs, miR-510 was further examined in another cohort of normal ovarian tissues, as well as the HGSC, low-grade serous carcinoma (LGSC) and CCC specimens using RT-qPCR and in situ hybridization. The results revealed that, of the 768 miRNAs analyzed in the microarray, 33 and 50 miRNAs were significantly upregulated and downregulated, respectively, with at least a 2-fold difference in HGSC, compared with CCC. The quantitative analysis demonstrated that miR-510 and miR-129-3p were significantly downregulated, and that miR-483-5p and miR-miR-449a were significantly upregulated in CCC, compared with HGSC (P<0.05), which was consistent with the microarray results. Kaplan-Meier analysis revealed low expression levels of miR-510 and low expression levels of miR-129-3p, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymphatic metastasis and that HGSC was significantly associated with the poorer overall survival rates (P<0.05). The expression of miR-510 was significantly higher in the LGSC and CCC tissues, compared with the HGSC and normal ovarian tissues. The results of the present study suggested that different subtypes of EOC have specific miRNA signatures, and that miR-510 may be involved differently in HGSC and CCC. Thus, miR-510 and miR-129-3p may be considered as potential novel candidate clinical biomarkers for predicting the outcome of EOC. PMID:26497752

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

PubMed

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions

PubMed Central

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity. PMID:25191551

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

PubMed

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah; Dallabernadina, Pietro; Boos, Irene; Andersen, Mathias C F; Kotake, Toshihisa; Knox, J Paul; Hahn, Michael G; Clausen, Mads H; Pfrengle, Fabian

2017-11-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision. © 2017 American Society of Plant Biologists. All Rights Reserved.

Analysis of Altered Micro RNA Expression Profiles in Focal Cortical Dysplasia IIB.

PubMed

Li, Lin; Liu, Chang-Qing; Li, Tian-Fu; Guan, Yu-Guang; Zhou, Jian; Qi, Xue-Ling; Yang, Yu-Tao; Deng, Jia-Hui; Xu, Zhi-Qing David; Luan, Guo-Ming

2016-04-01

Focal cortical dysplasia type IIB is a commonly encountered subtype of developmental malformation of the cerebral cortex and is often associated with pharmacoresistant epilepsy. In this study, to investigate the molecular etiology of focal cortical dysplasia type IIB, the authors performed micro ribonucleic acid (RNA) microarray on surgical specimens from 5 children (2 female and 3 male, mean age was 73.4 months, range 50-112 months) diagnosed of focal cortical dysplasia type IIB and matched normal tissue adjacent to the lesion. In all, 24 micro RNAs were differentially expressed in focal cortical dysplasia type IIB, and the microarray results were validated using quantitative real-time polymerase chain reaction (PCR). Then the putative target genes of the differentially expressed micro RNAs were identified by bioinformatics analysis. Moreover, biological significance of the target genes was evaluated by investigating the pathways in which the genes were enriched, and the Hippo signaling pathway was proposed to be highly related with the pathogenesis of focal cortical dysplasia type IIB. © The Author(s) 2015.

The effect of column purification on cDNA indirect labelling for microarrays

PubMed Central

Molas, M Lia; Kiss, John Z

2007-01-01

Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays. PMID:17597522

Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques.

PubMed

Khan, Ferdous; Tare, Rahul S; Kanczler, Janos M; Oreffo, Richard O C; Bradley, Mark

2010-03-01

A combination of high-throughput material formulation and microarray techniques were synergistically applied for the efficient analysis of the biological functionality of 135 binary polymer blends. This allowed the identification of cell-compatible biopolymers permissive for human skeletal stem cell growth in both in vitro and in vivo applications. The blended polymeric materials were developed from commercially available, inexpensive and well characterised biodegradable polymers, which on their own lacked both the structural requirements of a scaffold material and, critically, the ability to facilitate cell growth. Blends identified here proved excellent templates for cell attachment, and in addition, a number of blends displayed remarkable bone-like architecture and facilitated bone regeneration by providing 3D biomimetic scaffolds for skeletal cell growth and osteogenic differentiation. This study demonstrates a unique strategy to generate and identify innovative materials with widespread application in cell biology as well as offering a new reparative platform strategy applicable to skeletal tissues. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Cyclin D1 and Ewing's sarcoma/PNET: A microarray analysis.

PubMed

Fagone, Paolo; Nicoletti, Ferdinando; Salvatorelli, Lucia; Musumeci, Giuseppe; Magro, Gaetano

2015-10-01

Recent immunohistochemical analyses have showed that cyclin D1 is expressed in soft tissue Ewing's sarcoma/peripheral neuroectodermal tumor (PNET) of childhood and adolescents, while it is undetectable in both embryonal and alveolar rhabdomyosarcoma. In the present paper, microarray analysis provided evidence of a significant upregulation of cyclin D1 in Ewing's sarcoma as compared to normal tissues. In addition, we confirmed our previous findings of a significant over-expression of cyclin D1 in Ewing sarcoma as compared to rhabdomyosarcoma. Bioinformatic analysis also allowed to identify some other genes, strongly correlated to cyclin D1, which, although not previously studied in pediatric tumors, could represent novel markers for the diagnosis and prognosis of Ewing's sarcoma/PNET. The data herein provided support not only the use of cyclin D1 as a diagnostic marker of Ewing sarcoma/PNET but also the possibility of using drugs targeting cyclin D1 as potential therapeutic strategies. Copyright © 2015 Elsevier GmbH. All rights reserved.

Meta-review of protein network regulating obesity between validated obesity candidate genes in the white adipose tissue of high-fat diet-induced obese C57BL/6J mice.

PubMed

Kim, Eunjung; Kim, Eun Jung; Seo, Seung-Won; Hur, Cheol-Goo; McGregor, Robin A; Choi, Myung-Sook

2014-01-01

Worldwide obesity and related comorbidities are increasing, but identifying new therapeutic targets remains a challenge. A plethora of microarray studies in diet-induced obesity models has provided large datasets of obesity associated genes. In this review, we describe an approach to examine the underlying molecular network regulating obesity, and we discuss interactions between obesity candidate genes. We conducted network analysis on functional protein-protein interactions associated with 25 obesity candidate genes identified in a literature-driven approach based on published microarray studies of diet-induced obesity. The obesity candidate genes were closely associated with lipid metabolism and inflammation. Peroxisome proliferator activated receptor gamma (Pparg) appeared to be a core obesity gene, and obesity candidate genes were highly interconnected, suggesting a coordinately regulated molecular network in adipose tissue. In conclusion, the current network analysis approach may help elucidate the underlying molecular network regulating obesity and identify anti-obesity targets for therapeutic intervention.

Epigenetic transgenerational inheritance of somatic transcriptomes and epigenetic control regions

PubMed Central

2012-01-01

Background Environmentally induced epigenetic transgenerational inheritance of adult onset disease involves a variety of phenotypic changes, suggesting a general alteration in genome activity. Results Investigation of different tissue transcriptomes in male and female F3 generation vinclozolin versus control lineage rats demonstrated all tissues examined had transgenerational transcriptomes. The microarrays from 11 different tissues were compared with a gene bionetwork analysis. Although each tissue transgenerational transcriptome was unique, common cellular pathways and processes were identified between the tissues. A cluster analysis identified gene modules with coordinated gene expression and each had unique gene networks regulating tissue-specific gene expression and function. A large number of statistically significant over-represented clusters of genes were identified in the genome for both males and females. These gene clusters ranged from 2-5 megabases in size, and a number of them corresponded to the epimutations previously identified in sperm that transmit the epigenetic transgenerational inheritance of disease phenotypes. Conclusions Combined observations demonstrate that all tissues derived from the epigenetically altered germ line develop transgenerational transcriptomes unique to the tissue, but common epigenetic control regions in the genome may coordinately regulate these tissue-specific transcriptomes. This systems biology approach provides insight into the molecular mechanisms involved in the epigenetic transgenerational inheritance of a variety of adult onset disease phenotypes. PMID:23034163

Adipose tissue transcriptome changes during obesity development in female dogs.

PubMed

Grant, Ryan W; Vester Boler, Brittany M; Ridge, Tonya K; Graves, Thomas K; Swanson, Kelly S

2011-03-29

During the development of obesity, adipose tissue undergoes major expansion and remodeling, but the biological processes involved in this transition are not well understood. The objective of this study was to analyze global gene expression profiles of adipose tissue in dogs, fed a high-fat diet, during the transition from a lean to obese phenotype. Nine female beagles (4.09 ± 0.64 yr; 8.48 ± 0.35 kg) were randomized to ad libitum feeding or body weight maintenance. Subcutaneous adipose tissue biopsy, blood, and dual x-ray absorptiometry measurements were collected at 0, 4, 8, 12, and 24 wk of feeding. Serum was analyzed for glucose, insulin, fructosamine, triglycerides, free fatty acids, adiponectin, and leptin. Formalin-fixed adipose tissue was used for determination of adipocyte size. Adipose RNA samples were hybridized to Affymetrix Canine 2.0 microarrays. Statistical analysis, using repeated-measures ANOVA, showed ad libitum feeding increased (P < 0.05) body weight (0 wk, 8.36 ± 0.34 kg; 24 wk, 14.64 ± 0.34 kg), body fat mass (0 wk, 1.36 ± 0.24 kg; 24 wk, 6.52 ± 0.24 kg), adipocyte size (0 wk, 114.66 ± 17.38 μm(2); 24 wk, 320.97 ± 0.18.17 μm(2)), and leptin (0 wk, 0.8 ± 1.0 ng/ml; 24 wk, 12.9 ± 1.0 ng/ml). Microarrays displayed 1,665 differentially expressed genes in adipose tissue as weight increased. Alterations were seen in adipose tissue homeostatic processes including metabolism, oxidative stress, mitochondrial homeostasis, and extracellular matrix. Adipose transcriptome changes highlight the dynamic and adaptive response to ad libitum feeding and obesity development.

Tissue-specific mRNA expression profiling in grape berry tissues

PubMed Central

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. Conclusion These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality. PMID:17584945

MET amplification, expression, and exon 14 mutations in colorectal adenocarcinoma.

PubMed

Zhang, Meng; Li, Guichao; Sun, Xiangjie; Ni, Shujuan; Tan, Cong; Xu, Midie; Huang, Dan; Ren, Fei; Li, Dawei; Wei, Ping; Du, Xiang

2018-04-08

MET amplification, expression, and splice mutations at exon 14 result in dysregulation of the MET signaling pathway. The aim of this study was to identify the relationship between MET amplification, protein or mRNA expression, and mutations in colorectal cancer (CRC). MET immunohistochemistry (IHC) was used for MET protein expression analysis and fluorescence in situ hybridization (FISH) was used for MET amplification detection. Both analyses were performed in tissue microarrays (TMA) containing 294 of colorectal adenocarcinoma tissue samples and 131 samples of adjacent normal epithelial tissue. MET mRNA expression was examined by real-time quantitative polymerase chain reaction (qRT-PCR) in 72 fresh colorectal adenocarcinoma tissue samples and adjacent normal colon tissue. PCR sequencing was performed to screen for MET exon 14 splice mutations in 59 fresh CRC tissue samples. Our results showed that MET protein expression was higher in colorectal tumor tissue than in adjacent normal intestinal epithelium. Positive MET protein expression was associated with significantly poorer overall survival (OS) and disease-free survival (DFS). Multivariate analysis revealed that positive MET protein expression was an independent risk factor for DFS, but not for OS. MET mRNA expression was upregulated in tumor tissues compared with the adjacent normal tissues. The incidence of MET amplification was 4.4%. None of the patients was positive for MET mutation. Collectively, MET was overexpressed in colorectal adenocarcinoma, and its positive protein expression predicted a poorer outcome in CRC patients. Furthermore, according to our results, MET amplification and 14 exon mutation are extremely rare events in colorectal adenocarcinoma. Copyright © 2018. Published by Elsevier Inc.

Gene expression profiles help identify the Tissue of Origin for metastatic brain cancers

PubMed Central

2010-01-01

Background Metastatic brain cancers are the most common intracranial tumor and occur in about 15% of all cancer patients. In up to 10% of these patients, the primary tumor tissue remains unknown, even after a time consuming and costly workup. The Pathwork® Tissue of Origin Test (Pathwork Diagnostics, Redwood City, CA, USA) is a gene expression test to aid in the diagnosis of metastatic, poorly differentiated and undifferentiated tumors. It measures the expression pattern of 1,550 genes in these tumors and compares it to the expression pattern of a panel of 15 known tumor types. The purpose of this study was to evaluate the performance of the Tissue of Origin Test in the diagnosis of primary sites for metastatic brain cancer patients. Methods Fifteen fresh-frozen metastatic brain tumor specimens of known origins met specimen requirements. These specimens were entered into the study and processed using the Tissue of Origin Test. Results were compared to the known primary site and the agreement between the two results was assessed. Results Fourteen of the fifteen specimens produced microarray data files that passed all quality metrics. One originated from a tissue type that was off-panel. Among the remaining 13 cases, the Tissue of Origin Test accurately predicted the available diagnosis in 12/13 (92.3%) cases. Discussion This study demonstrates the accuracy of the Tissue of Origin Test when applied to predict the tissue of origin of metastatic brain tumors. This test could be a very useful tool for pathologists as they classify metastatic brain cancers. PMID:20420692

Discovery of Colorectal Cancer Biomarker Candidates by Membrane Proteomic Analysis and Subsequent Verification using Selected Reaction Monitoring (SRM) and Tissue Microarray (TMA) Analysis*

PubMed Central

Kume, Hideaki; Muraoka, Satoshi; Kuga, Takahisa; Adachi, Jun; Narumi, Ryohei; Watanabe, Shio; Kuwano, Masayoshi; Kodera, Yoshio; Matsushita, Kazuyuki; Fukuoka, Junya; Masuda, Takeshi; Ishihama, Yasushi; Matsubara, Hisahiro; Nomura, Fumio; Tomonaga, Takeshi

2014-01-01

Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n = 10), cancer without metastasis (n = 10), cancer with metastasis (n = 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851. PMID:24687888

Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis.

PubMed

Kume, Hideaki; Muraoka, Satoshi; Kuga, Takahisa; Adachi, Jun; Narumi, Ryohei; Watanabe, Shio; Kuwano, Masayoshi; Kodera, Yoshio; Matsushita, Kazuyuki; Fukuoka, Junya; Masuda, Takeshi; Ishihama, Yasushi; Matsubara, Hisahiro; Nomura, Fumio; Tomonaga, Takeshi

2014-06-01

Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n = 10), cancer without metastasis (n = 10), cancer with metastasis (n = 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Too much data, but little inter-changeability: a lesson learned from mining public data on tissue specificity of gene expression.

PubMed

Li, Shuyu; Li, Yiqun Helen; Wei, Tao; Su, Eric Wen; Duffin, Kevin; Liao, Birong

2006-10-25

The tissue expression pattern of a gene often provides an important clue to its potential role in a biological process. A vast amount of gene expression data have been and are being accumulated in public repository through different technology platforms. However, exploitations of these rich data sources remain limited in part due to issues of technology standardization. Our objective is to test the data comparability between SAGE and microarray technologies, through examining the expression pattern of genes under normal physiological states across variety of tissues. There are 42-54% of genes showing significant correlations in tissue expression patterns between SAGE and GeneChip, with 30-40% of genes whose expression patterns are positively correlated and 10-15% of genes whose expression patterns are negatively correlated at a statistically significant level (p = 0.05). Our analysis suggests that the discrepancy on the expression patterns derived from technology platforms is not likely from the heterogeneity of tissues used in these technologies, or other spurious correlations resulting from microarray probe design, abundance of genes, or gene function. The discrepancy can be partially explained by errors in the original assignment of SAGE tags to genes due to the evolution of sequence databases. In addition, sequence analysis has indicated that many SAGE tags and Affymetrix array probe sets are mapped to different splice variants or different sequence regions although they represent the same gene, which also contributes to the observed discrepancies between SAGE and array expression data. To our knowledge, this is the first report attempting to mine gene expression patterns across tissues using public data from different technology platforms. Unlike previous similar studies that only demonstrated the discrepancies between the two gene expression platforms, we carried out in-depth analysis to further investigate the cause for such discrepancies. Our study shows that the exploitation of rich public expression resource requires extensive knowledge about the technologies, and experiment. Informatic methodologies for better interoperability among platforms still remain a gap. One of the areas that can be improved practically is the accurate sequence mapping of SAGE tags and array probes to full-length genes.

Hormonal receptors and vascular endothelial growth factor in juvenile nasopharyngeal angiofibroma: immunohistochemical and tissue microarray analysis.

PubMed

Liu, Zhuofu; Wang, Jingjing; Wang, Huan; Wang, Dehui; Hu, Li; Liu, Quan; Sun, Xicai

2015-01-01

This work demonstrated that juvenile nasopharyngeal angiofibromas (JNAs) express high levels of hormone receptors and vascular endothelial growth factor (VEGF) compared with normal nasal mucosa. The interaction between hormone receptors and VEGF may be involved in the initiation and growth of JNA. JNA is a rare benign tumor that occurs almost exclusively in male adolescents. Although generally regarded as a hormone-dependent tumor, this has not been proven in previous studies. The aim of this study was to investigate the role of hormone receptors in JNA and the relationship with clinical characteristics. Standard immunohistochemical microarray analysis was performed on 70 JNA samples and 10 turbinate tissue samples. Specific antibodies for androgen receptor (AR), estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), progesterone receptor (PR), and VEGF were examined, and the relationships of receptor expression with age, tumor stage, and bleeding were evaluated. RESULTS showed that JNA expressed ER-α (92.9%), ER-β (91.4%), AR (65.7%), PR (12.8%), and VEGF (95.7%) at different levels. High level of VEGF was linked to elevated ER-α and ER-β. There was no significant relationship between hormonal receptors and age at diagnosis, tumor stage or bleeding. However, overexpression of ER-α was found to be an indicator of poor prognosis (p = 0.031).

[Theoretical foundations of protein chips and their possible use in medical research and diagnostics].

PubMed

Spisák, Sándor; Molnár, Béla; Galamb, Orsolya; Sipos, Ferenc; Tulassay, Zsolt

2007-08-12

The confirmation of mRNA expression studies by protein chips is of high recent interest due to the widespread application of expression arrays. In this review the advantages, technical limitations, application fields and the first results of the protein arrays is described. The bottlenecks of the increasing protein array applications are the fast decomposition of proteins, the problem with aspecific binding and the lack of amplification techniques. Today glass slide based printed, SELDI (MS) based, electrophoresis based and tissue microarray based technologies are available. The advantage of the glass slide based chips are the simplicity of their application, and relatively low cost. The SELDI based protein chip technique is applicable to minute amounts of starting material (<1 microg) but it is the most expensive one. The electrophoresis based techniques are still under intensive development. The tissue microarrays can be used for the parallel testing of the sensitivity and specificity of single antibodies on a broad range of histological specimens on a single slide. Protein chips were successfully used for serum tumor marker detection, cancer research, cell physiology studies and for the verification of mRNA expression studies. Protein chips are envisioned to be available for routine diagnostic applications if the ongoing technology development will be successful in increase in sensitivity, specificity, costs reduction and for the reduction of the necessary sample volume.

Generation of Viable Cell and Biomaterial Patterns by Laser Transfer

NASA Astrophysics Data System (ADS)

Ringeisen, Bradley

2001-03-01

In order to fabricate and interface biological systems for next generation applications such as biosensors, protein recognition microarrays, and engineered tissues, it is imperative to have a method of accurately and rapidly depositing different active biomaterials in patterns or layered structures. Ideally, the biomaterial structures would also be compatible with many different substrates including technologically relevant platforms such as electronic circuits or various detection devices. We have developed a novel laser-based technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW), that is able to direct write patterns and three-dimensional structures of numerous biologically active species ranging from proteins and antibodies to living cells. Specifically, we have shown that MAPLE DW is capable of forming mesoscopic patterns of living prokaryotic cells (E. coli bacteria), living mammalian cells (Chinese hamster ovaries), active proteins (biotinylated bovine serum albumin, horse radish peroxidase), and antibodies specific to a variety of classes of cancer related proteins including intracellular and extracellular matrix proteins, signaling proteins, cell cycle proteins, growth factors, and growth factor receptors. In addition, patterns of viable cells and active biomolecules were deposited on different substrates including metals, semiconductors, nutrient agar, and functionalized glass slides. We will present an explanation of the laser-based transfer mechanism as well as results from our recent efforts to fabricate protein recognition microarrays and tissue-based microfluidic networks.

Clearance of a persistent Picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

USDA-ARS?s Scientific Manuscript database

Immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in distinct microanatomic compartments of the bovine nasopharynx were investigated using quantitative RT-PCR and whole transcriptome microarray. Analysis of tissue samples obtained during ...

Identification of Gender-specific Transcripts by Microarray in Gonad Tissue of Larval and Juvenile Xenopus tropicalis

EPA Science Inventory

Amphibian model species Xenopus tropicalis is currently being utilized by EPA in the development of a standardized in vivo reproductive toxicity assay. Perturbations to the hypothalamic-pituitary-gonadal axis from exposure to endocrine disrupting compounds during larval develop...

Transcriptome response to elevated CO2, water deficit, and thermal stress in peanut

USDA-ARS?s Scientific Manuscript database

Previously, our laboratories have performed gene expression studies using EST sequencing and spotted microarrays to investigate tissue-specific gene expression and response to abiotic stress. While these studies have provided valuable insight into these processes, they are constrained by sequencer t...

Use of a 15 k gene microarray to determine gene expression changes in response to acute and chronic methylmercury exposure in the fathead minnow Pimephales promelas Rafinesque

USGS Publications Warehouse

Klaper, R.; Carter, Barbara J.; Richter, C.A.; Drevnick, P.E.; Sandheinrich, M.B.; Tillitt, D.E.

2008-01-01

This study describes the use of a 15 000 gene microarray developed for the toxicological model species, Pimephales promelas, in investigating the impact of acute and chronic methylmercury exposures in male gonad and liver tissues. The results show significant differences in the individual genes that were differentially expressed in response to each treatment. In liver, a total of 650 genes exhibited significantly (P < 0.05) altered expression with greater than two-fold differences from the controls in response to acute exposure and a total of 267 genes were differentially expressed in response to chronic exposure. A majority of these genes were downregulated rather than upregulated. Fewer genes were altered in gonad than in liver at both timepoints. A total of 212 genes were differentially expressed in response to acute exposure and 155 genes were altered in response to chronic exposure. Despite the differences in individual genes expressed across treatments, the functional categories that altered genes were associated with showed some similarities. Of interest in light of other studies involving the effects of methylmercury on fish, several genes associated with apoptosis were upregulated in response to both acute and chronic exposures. Induction of apoptosis has been associated with effects on reproduction seen in the previous studies. This study demonstrates the utility of microarray analysis for investigations of the physiological effects of toxicants as well as the time-course of effects that may take place. In addition, it is the first publication to demonstrate the use of this new 15 000 gene microarray for fish biology and toxicology. ?? 2008 The Authors.

Near-isogenic cotton germplasm lines that differ in fiber-bundle strength have temporal differences in fiber gene expression patterns as revealed by comparative high-throughput profiling.

PubMed

Hinchliffe, Doug J; Meredith, William R; Yeater, Kathleen M; Kim, Hee Jin; Woodward, Andrew W; Chen, Z Jeffrey; Triplett, Barbara A

2010-05-01

Gene expression profiles of developing cotton (Gossypium hirsutum L.) fibers from two near-isogenic lines (NILs) that differ in fiber-bundle strength, short-fiber content, and in fewer than two genetic loci were compared using an oligonucleotide microarray. Fiber gene expression was compared at five time points spanning fiber elongation and secondary cell wall (SCW) biosynthesis. Fiber samples were collected from field plots in a randomized, complete block design, with three spatially distinct biological replications for each NIL at each time point. Microarray hybridizations were performed in a loop experimental design that allowed comparisons of fiber gene expression profiles as a function of time between the two NILs. Overall, developmental expression patterns revealed by the microarray experiment agreed with previously reported cotton fiber gene expression patterns for specific genes. Additionally, genes expressed coordinately with the onset of SCW biosynthesis in cotton fiber correlated with gene expression patterns of other SCW-producing plant tissues. Functional classification and enrichment analysis of differentially expressed genes between the two NILs revealed that genes associated with SCW biosynthesis were significantly up-regulated in fibers of the high-fiber quality line at the transition stage of cotton fiber development. For independent corroboration of the microarray results, 15 genes were selected for quantitative reverse transcription PCR analysis of fiber gene expression. These analyses, conducted over multiple field years, confirmed the temporal difference in fiber gene expression between the two NILs. We hypothesize that the loci conferring temporal differences in fiber gene expression between the NILs are important regulatory sequences that offer the potential for more targeted manipulation of cotton fiber quality.

A preliminary study of differentially expressed genes in expanded skin and normal skin: implications for adult skin regeneration.

PubMed

Yang, Mei; Liang, Yimin; Sheng, Lingling; Shen, Guoxiong; Liu, Kai; Gu, Bin; Meng, Fanjun; Li, Qingfeng

2011-03-01

In adults, severely damaged skin heals by scar formation and cannot regenerate to the original skin structure. However, tissue expansion is an exception, as normal skin regenerates under the mechanical stretch resulting from tissue expansion. This technique has been used clinically for defect repair and organ reconstruction for decades. However, the phenomenon of adult skin regeneration during tissue expansion has caused little attention, and the mechanism of skin regeneration during tissue expansion has not been fully understood. In this study, microarray analysis was performed on expanded human skin and normal human skin. Significant difference was observed in 77 genes, which suggest a network of several integrated cascades, including cytokines, extracellular, cytoskeletal, transmembrane molecular systems, ion or ion channels, protein kinases and transcriptional systems, is involved in the skin regeneration during expansion. Among these, the significant expression of some regeneration related genes, such as HOXA5, HOXB2 and AP1, was the first report in tissue expansion. Data in this study suggest a list of candidate genes, which may help to elucidate the fundamental mechanism of skin regeneration during tissue expansion and which may have implications for postnatal skin regeneration and therapeutic interventions in wound healing.

The effect of column purification on cDNA indirect labelling for microarrays.

PubMed

Molas, M Lia; Kiss, John Z

2007-06-27

The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays.

SYMBIOmatics: synergies in Medical Informatics and Bioinformatics--exploring current scientific literature for emerging topics.

PubMed

Rebholz-Schuhman, Dietrich; Cameron, Graham; Clark, Dominic; van Mulligen, Erik; Coatrieux, Jean-Louis; Del Hoyo Barbolla, Eva; Martin-Sanchez, Fernando; Milanesi, Luciano; Porro, Ivan; Beltrame, Francesco; Tollis, Ioannis; Van der Lei, Johan

2007-03-08

The SYMBIOmatics Specific Support Action (SSA) is "an information gathering and dissemination activity" that seeks "to identify synergies between the bioinformatics and the medical informatics" domain to improve collaborative progress between both domains (ref. to http://www.symbiomatics.org). As part of the project experts in both research fields will be identified and approached through a survey. To provide input to the survey, the scientific literature was analysed to extract topics relevant to both medical informatics and bioinformatics. This paper presents results of a systematic analysis of the scientific literature from medical informatics research and bioinformatics research. In the analysis pairs of words (bigrams) from the leading bioinformatics and medical informatics journals have been used as indication of existing and emerging technologies and topics over the period 2000-2005 ("recent") and 1990-1990 ("past"). We identified emerging topics that were equally important to bioinformatics and medical informatics in recent years such as microarray experiments, ontologies, open source, text mining and support vector machines. Emerging topics that evolved only in bioinformatics were system biology, protein interaction networks and statistical methods for microarray analyses, whereas emerging topics in medical informatics were grid technology and tissue microarrays. We conclude that although both fields have their own specific domains of interest, they share common technological developments that tend to be initiated by new developments in biotechnology and computer science.

Comprehensive analysis of differentially expressed profiles of lncRNAs and construction of miR-133b mediated ceRNA network in colorectal cancer.

PubMed

Wu, Hao; Wu, Runliu; Chen, Miao; Li, Daojiang; Dai, Jing; Zhang, Yi; Gao, Kai; Yu, Jun; Hu, Gui; Guo, Yihang; Lin, Changwei; Li, Xiaorong

2017-03-28

Growing evidence suggests that long non-coding RNAs (lncRNAs) play a key role in tumorigenesis. However, the mechanism remains largely unknown. Thousands of significantly dysregulated lncRNAs and mRNAs were identified by microarray. Furthermore, a miR-133b-meditated lncRNA-mRNA ceRNA network was revealed, a subset of which was validated in 14 paired CRC patient tumor/non-tumor samples. Gene set enrichment analysis (GSEA) results demonstrated that lncRNAs ENST00000520055 and ENST00000535511 shared KEGG pathways with miR-133b target genes. We used microarrays to survey the lncRNA and mRNA expression profiles of colorectal cancer and para-cancer tissues. Gene Ontology (GO) and KEGG pathway enrichment analyses were performed to explore the functions of the significantly dysregulated genes. An innovate method was employed that combined analyses of two microarray data sets to construct a miR-133b-mediated lncRNA-mRNA competing endogenous RNAs (ceRNA) network. Quantitative RT-PCR analysis was used to validate part of this network. GSEA was used to predict the potential functions of these lncRNAs. This study identifies and validates a new method to investigate the miR-133b-mediated lncRNA-mRNA ceRNA network and lays the foundation for future investigation into the role of lncRNAs in colorectal cancer.

The Genome-Wide Expression Profile of Saussurea lappa Extract on House Dust Mite-Induced Atopic Dermatitis in Nc/Nga Mice.

PubMed

Lim, Hye-Sun; Ha, Hyekyung; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

2015-09-01

Saussurea lappa has been reported to possess anti-atopic properties. In this study, we have confirmed the S. lappa's anti-atopic properties in Nc/Nga mice and investigated the candidate gene related with its properties using microarray. We determined the target gene using real time PCR in in vitro experiment. S. lappa showed the significant reduction in atopic dermatitis (AD) score and immunoglobulin E compared with the AD induced Nc/Nga mice. In the results of microarray using back skin obtained from animals, we found that S. lappa's properties are closely associated with cytokine-cytokine receptor interaction and the JAK-STAT signaling pathway. Consistent with the microarray data, real-time RT-PCR confirmed these modulation at the mRNA level in skin tissues from S. lappa-treated mice. Among these genes, PI3Kca and IL20Rβ were significantly downregulated by S. lappa treatment in Nc/Nga mouse model. In in vitro experiment using HaCaT cells, we found that the S. lappa components, including alantolactone, caryophyllene, costic acid, costunolide and dehydrocostus lactone significantly decreased the expression of PI3Kca but not IL20Rβ in vitro. Therefore, our study suggests that PI3Kca-related signaling is closely related with the protective effects of S. lappa against the development of atopic-dermatitis.

Customizing chemotherapy for colon cancer: the potential of gene expression profiling.

PubMed

Mariadason, John M; Arango, Diego; Augenlicht, Leonard H

2004-06-01

The value of gene expression profiling, or microarray analysis, for the classification and prognosis of multiple forms of cancer is now clearly established. For colon cancer, expression profiling can readily discriminate between normal and tumor tissue, and to some extent between tumors of different histopathological stage and prognosis. While a definitive in vivo study demonstrating the potential of this methodology for predicting response to chemotherapy is presently lacking, the ability of microarrays to distinguish other subtleties of colon cancer phenotype, as well as recent in vitro proof-of-principle experiments utilizing colon cancer cell lines, illustrate the potential of this methodology for predicting the probability of response to specific chemotherapeutic agents. This review discusses some of the recent advances in the use of microarray analysis for understanding and distinguishing colon cancer subtypes, and attempts to identify challenges that need to be overcome in order to achieve the goal of using gene expression profiling for customizing chemotherapy in colon cancer.

Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

PubMed Central

Wang, Denong; Tang, Jin; Liu, Shaoyi

2015-01-01

Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gpHAE3, was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation. PMID:26539555

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

Analysis of Temporal-spatial Co-variation within Gene Expression Microarray Data in an Organogenesis Model

NASA Astrophysics Data System (ADS)

Ehler, Martin; Rajapakse, Vinodh; Zeeberg, Barry; Brooks, Brian; Brown, Jacob; Czaja, Wojciech; Bonner, Robert F.

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. We used a novel clustering method based on Laplacian Eigenmaps, a nonlinear dimension reduction method, to analyze microarray data from laser capture microdissected (LCM) cells at the site and developmental stages (days 10.5 to 12.5) of optic fissure closure. Our new method provided greater biological specificity than classical clustering algorithms in terms of identifying more biological processes and functions related to eye development as defined by Gene Ontology at lower false discovery rates. This new methodology builds on the advantages of LCM to isolate pure phenotypic populations within complex tissues and allows improved ability to identify critical gene products expressed at lower copy number. The combination of LCM of embryonic organs, gene expression microarrays, and extracting spatial and temporal co-variations appear to be a powerful approach to understanding the gene regulatory networks that specify mammalian organogenesis.

LncRNA-FEZF1-AS1 promotes tumor proliferation and metastasis in colorectal cancer by regulating PKM2 signaling.

PubMed

Bian, Zehua; Zhang, Jiwei; Li, Min; Feng, Yuyang; Wang, Xue; Zhang, Jia; Yao, Surui; Jin, Guoying; Du, Jun; Han, Weifeng; Yin, Yuan; Huang, Shenglin; Fei, Bojian; Zou, Jian; Huang, Zhaohui

2018-06-18

Long non-coding RNAs (lncRNAs) play key roles in human cancers. Here, FEZF1-AS1, a highly overexpressed lncRNA in colorectal cancer (CRC), was identified by lncRNA microarrays. We aimed to explore the roles and possible molecular mechanisms of FEZF1-AS1 in CRC. LncRNA expression in CRC tissues was measured by lncRNA microarray and qRT-PCR. The functional roles of FEZF1-AS1 in CRC were demonstrated by a series of in vitro and in vivo experiments. RNA pull-down, RNA immunoprecipitation and luciferase analyses were used to demonstrate the potential mechanisms of FEZF1-AS1. We identified a series of differentially expressed lncRNAs in CRC using lncRNA microarrays, and revealed that FEZF1-AS1 is one of the most overexpressed. Further validation in two expanded CRC cohorts confirmed the upregulation of FEZF1-AS1 in CRC, and revealed that increased FEZF1-AS1 expression is associated with poor survival. Functional assays revealed that FEZF1-AS1 promotes CRC cell proliferation and metastasis. Mechanistically, FEZF1-AS1 could bind and increase the stability of the pyruvate kinase 2 (PKM2) protein, resulting in increased cytoplasmic and nuclear PKM2 levels. Increased cytoplasmic PKM2 promoted pyruvate kinase activity and lactate production (aerobic glycolysis), whereas FEZF1-AS1-induced nuclear PKM2 upregulation further activated STAT3 signaling. In addition, PKM2 was upregulated in CRC tissues and correlated with FEZF1-AS1 expression and patient survival. Together, these data provide mechanistic insights into the regulation of FEZF1-AS1 on both STAT3 signaling and glycolysis by binding PKM2 and increasing its stability. Copyright ©2018, American Association for Cancer Research.

Probing the Xenopus laevis inner ear transcriptome for biological function

PubMed Central

2012-01-01

Background The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus Xenopus is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the Xenopus laevis inner ear transcriptome through microarray analysis. Results Microarray analysis uncovered genes within the X. laevis inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of Xenopus in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the Affymetrix GeneChip® Xenopus laevis Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization. Conclusions We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the Xenopus inner ear transcriptome, thereby addressing the impediment imposed by insufficient gene annotation. These findings heighten the relevance of Xenopus as a model organism for genetic investigations of inner ear organogenesis, morphogenesis, and regeneration. PMID:22676585

Detecting discordance enrichment among a series of two-sample genome-wide expression data sets.

PubMed

Lai, Yinglei; Zhang, Fanni; Nayak, Tapan K; Modarres, Reza; Lee, Norman H; McCaffrey, Timothy A

2017-01-25

With the current microarray and RNA-seq technologies, two-sample genome-wide expression data have been widely collected in biological and medical studies. The related differential expression analysis and gene set enrichment analysis have been frequently conducted. Integrative analysis can be conducted when multiple data sets are available. In practice, discordant molecular behaviors among a series of data sets can be of biological and clinical interest. In this study, a statistical method is proposed for detecting discordance gene set enrichment. Our method is based on a two-level multivariate normal mixture model. It is statistically efficient with linearly increased parameter space when the number of data sets is increased. The model-based probability of discordance enrichment can be calculated for gene set detection. We apply our method to a microarray expression data set collected from forty-five matched tumor/non-tumor pairs of tissues for studying pancreatic cancer. We divided the data set into a series of non-overlapping subsets according to the tumor/non-tumor paired expression ratio of gene PNLIP (pancreatic lipase, recently shown it association with pancreatic cancer). The log-ratio ranges from a negative value (e.g. more expressed in non-tumor tissue) to a positive value (e.g. more expressed in tumor tissue). Our purpose is to understand whether any gene sets are enriched in discordant behaviors among these subsets (when the log-ratio is increased from negative to positive). We focus on KEGG pathways. The detected pathways will be useful for our further understanding of the role of gene PNLIP in pancreatic cancer research. Among the top list of detected pathways, the neuroactive ligand receptor interaction and olfactory transduction pathways are the most significant two. Then, we consider gene TP53 that is well-known for its role as tumor suppressor in cancer research. The log-ratio also ranges from a negative value (e.g. more expressed in non-tumor tissue) to a positive value (e.g. more expressed in tumor tissue). We divided the microarray data set again according to the expression ratio of gene TP53. After the discordance enrichment analysis, we observed overall similar results and the above two pathways are still the most significant detections. More interestingly, only these two pathways have been identified for their association with pancreatic cancer in a pathway analysis of genome-wide association study (GWAS) data. This study illustrates that some disease-related pathways can be enriched in discordant molecular behaviors when an important disease-related gene changes its expression. Our proposed statistical method is useful in the detection of these pathways. Furthermore, our method can also be applied to genome-wide expression data collected by the recent RNA-seq technology.

Equalizer reduces SNP bias in Affymetrix microarrays.

PubMed

Quigley, David

2015-07-30

Gene expression microarrays measure the levels of messenger ribonucleic acid (mRNA) in a sample using probe sequences that hybridize with transcribed regions. These probe sequences are designed using a reference genome for the relevant species. However, most model organisms and all humans have genomes that deviate from their reference. These variations, which include single nucleotide polymorphisms, insertions of additional nucleotides, and nucleotide deletions, can affect the microarray's performance. Genetic experiments comparing individuals bearing different population-associated single nucleotide polymorphisms that intersect microarray probes are therefore subject to systemic bias, as the reduction in binding efficiency due to a technical artifact is confounded with genetic differences between parental strains. This problem has been recognized for some time, and earlier methods of compensation have attempted to identify probes affected by genome variants using statistical models. These methods may require replicate microarray measurement of gene expression in the relevant tissue in inbred parental samples, which are not always available in model organisms and are never available in humans. By using sequence information for the genomes of organisms under investigation, potentially problematic probes can now be identified a priori. However, there is no published software tool that makes it easy to eliminate these probes from an annotation. I present equalizer, a software package that uses genome variant data to modify annotation files for the commonly used Affymetrix IVT and Gene/Exon platforms. These files can be used by any microarray normalization method for subsequent analysis. I demonstrate how use of equalizer on experiments mapping germline influence on gene expression in a genetic cross between two divergent mouse species and in human samples significantly reduces probe hybridization-induced bias, reducing false positive and false negative findings. The equalizer package reduces probe hybridization bias from experiments performed on the Affymetrix microarray platform, allowing accurate assessment of germline influence on gene expression.

MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

PubMed

Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

2012-10-15

Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

Oncogene GAEC1 regulates CAPN10 expression which predicts survival in esophageal squamous cell carcinoma

PubMed Central

Chan, Dessy; Tsoi, Miriam Yuen-Tung; Liu, Christina Di; Chan, Sau-Hing; Law, Simon Ying-Kit; Chan, Kwok-Wah; Chan, Yuen-Piu; Gopalan, Vinod; Lam, Alfred King-Yin; Tang, Johnny Cheuk-On

2013-01-01

AIM: To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance. METHODS: The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses. RESULTS: The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16.33 vs 12.62 ± 12.44, P = 0.032). No significant correction was observed among the TNRC6C protein expression level and the clinocopathologcial features of esophageal squamous cell carcinoma. CONCLUSION: GAEC1 regulates the expression of CAPN10 and TNRC6C downstream. Calpain 10 expression is a potential prognostic marker in patients with esophageal squamous cell carcinoma. PMID:23687414

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome

PubMed Central

2010-01-01

Background Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease. PMID:20929581

Identifying significant genetic regulatory networks in the prostate cancer from microarray data based on transcription factor analysis and conditional independency.

PubMed

Yeh, Hsiang-Yuan; Cheng, Shih-Wu; Lin, Yu-Chun; Yeh, Cheng-Yu; Lin, Shih-Fang; Soo, Von-Wun

2009-12-21

Prostate cancer is a world wide leading cancer and it is characterized by its aggressive metastasis. According to the clinical heterogeneity, prostate cancer displays different stages and grades related to the aggressive metastasis disease. Although numerous studies used microarray analysis and traditional clustering method to identify the individual genes during the disease processes, the important gene regulations remain unclear. We present a computational method for inferring genetic regulatory networks from micorarray data automatically with transcription factor analysis and conditional independence testing to explore the potential significant gene regulatory networks that are correlated with cancer, tumor grade and stage in the prostate cancer. To deal with missing values in microarray data, we used a K-nearest-neighbors (KNN) algorithm to determine the precise expression values. We applied web services technology to wrap the bioinformatics toolkits and databases to automatically extract the promoter regions of DNA sequences and predicted the transcription factors that regulate the gene expressions. We adopt the microarray datasets consists of 62 primary tumors, 41 normal prostate tissues from Stanford Microarray Database (SMD) as a target dataset to evaluate our method. The predicted results showed that the possible biomarker genes related to cancer and denoted the androgen functions and processes may be in the development of the prostate cancer and promote the cell death in cell cycle. Our predicted results showed that sub-networks of genes SREBF1, STAT6 and PBX1 are strongly related to a high extent while ETS transcription factors ELK1, JUN and EGR2 are related to a low extent. Gene SLC22A3 may explain clinically the differentiation associated with the high grade cancer compared with low grade cancer. Enhancer of Zeste Homolg 2 (EZH2) regulated by RUNX1 and STAT3 is correlated to the pathological stage. We provide a computational framework to reconstruct the genetic regulatory network from the microarray data using biological knowledge and constraint-based inferences. Our method is helpful in verifying possible interaction relations in gene regulatory networks and filtering out incorrect relations inferred by imperfect methods. We predicted not only individual gene related to cancer but also discovered significant gene regulation networks. Our method is also validated in several enriched published papers and databases and the significant gene regulatory networks perform critical biological functions and processes including cell adhesion molecules, androgen and estrogen metabolism, smooth muscle contraction, and GO-annotated processes. Those significant gene regulations and the critical concept of tumor progression are useful to understand cancer biology and disease treatment.

A Self-Directed Method for Cell-Type Identification and Separation of Gene Expression Microarrays

PubMed Central

Zuckerman, Neta S.; Noam, Yair; Goldsmith, Andrea J.; Lee, Peter P.

2013-01-01

Gene expression analysis is generally performed on heterogeneous tissue samples consisting of multiple cell types. Current methods developed to separate heterogeneous gene expression rely on prior knowledge of the cell-type composition and/or signatures - these are not available in most public datasets. We present a novel method to identify the cell-type composition, signatures and proportions per sample without need for a-priori information. The method was successfully tested on controlled and semi-controlled datasets and performed as accurately as current methods that do require additional information. As such, this method enables the analysis of cell-type specific gene expression using existing large pools of publically available microarray datasets. PMID:23990767

Lack of concordance in microarray gene expression responses to Phenobarbital in companion aged FFPE and Frozen liver samples

EPA Science Inventory

Despite the immense potential value of public and private biorepositories, direct utilization of archival tissues for molecular profiling has been limited. A major reason for this limited use is the difficulty in obtaining reliable transcriptomic profiles from formalin-fixed par...

Analysis of molecular pathways in pancreatic ductal adenocarcinomas with a bioinformatics approach.

PubMed

Wang, Yan; Li, Yan

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death worldwide. Our study aimed to reveal molecular mechanisms. Microarray data of GSE15471 (including 39 matching pairs of pancreatic tumor tissues and patient-matched normal tissues) was downloaded from Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs) in PDAC tissues compared with normal tissues by limma package in R language. Then GO and KEGG pathway enrichment analyses were conducted with online DAVID. In addition, principal component analysis was performed and a protein-protein interaction network was constructed to study relationships between the DEGs through database STRING. A total of 532 DEGs were identified in the 38 PDAC tissues compared with 33 normal tissues. The results of principal component analysis of the top 20 DEGs could differentiate the PDAC tissues from normal tissues directly. In the PPI network, 8 of the 20 DEGs were all key genes of the collagen family. Additionally, FN1 (fibronectin 1) was also a hub node in the network. The genes of the collagen family as well as FN1 were significantly enriched in complement and coagulation cascades, ECM-receptor interaction and focal adhesion pathways. Our results suggest that genes of collagen family and FN1 may play an important role in PDAC progression. Meanwhile, these DEGs and enriched pathways, such as complement and coagulation cascades, ECM-receptor interaction and focal adhesion may be important molecular mechanisms involved in the development and progression of PDAC.

MICROARRAY QUALITY CONTROL PROJECT: A COMPREHENSIVE GENE EXPRESSION TECHNOLOGY SURVEY DEMONSTRATES MEASURABLE CONSISTENCY AND CONCORDANT RESULTS BETWEEN PLATFORMS

EPA Science Inventory

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, h...

Profiling of the yak skeletal muscle tissue gene expression and comparison with the domestic cattle by genome array.

PubMed

Wang, H B; Zan, L S; Zhang, Y Y

2014-01-01

Of all the mammals of the world, the yak lives at the highest altitude area of more than 3000 m. Comparison between yak and cattle of the low-altitude areas will be informative in studying animal adaptation to higher altitudes. To investigate the molecular mechanism involved in meat quality differences between the two Chinese special varieties Qinghai yak and Qinchuan cattle, 12 chemical-physical characteristics of the longissimus dorsi muscle related to meat quality were compared at the age of 36 months, and the gene expression profiles were constructed by utilizing the bovine genome array. Significant analysis of microarrays was used to identify the differentially expressed genes. Gene ontology and pathway analysis were performed by a free Web-based Molecular Annotation System 2.0. The results reveal ~11 000 probes representing about 10 000 genes that were detected in both the Qinghai yak and Qinchuan cattle. A total of 1922 genes were shown to be differentially expressed, 633 probes were upregulated and 1259 probes were downregulated in the muscle tissue of Qinghai yak that were mainly involved in ubiquitin-mediated proteolysis, muscle growth regulation, glucose metabolism, immune response and so on. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. Further analysis implied that animals living at a high altitude may supply energy by more active protein catabolism and glycolysis compared with those living in the plain areas. Our results establish the groundwork for further studies on yaks' meat quality and will be beneficial in improving the yaks' breeding by molecular biotechnology.

The 'PUCE CAFE' Project: the first 15K coffee microarray, a new tool for discovering candidate genes correlated to agronomic and quality traits.

PubMed

Privat, Isabelle; Bardil, Amélie; Gomez, Aureliano Bombarely; Severac, Dany; Dantec, Christelle; Fuentes, Ivanna; Mueller, Lukas; Joët, Thierry; Pot, David; Foucrier, Séverine; Dussert, Stéphane; Leroy, Thierry; Journot, Laurent; de Kochko, Alexandre; Campa, Claudine; Combes, Marie-Christine; Lashermes, Philippe; Bertrand, Benoit

2011-01-05

Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

Delayed inflammatory mRNA and protein expression after spinal cord injury

PubMed Central

2011-01-01

Background Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. Methods Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. Results Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. Conclusions These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss. PMID:21975064

miRNAs in human subcutaneous adipose tissue: Effects of weight loss induced by hypocaloric diet and exercise.

PubMed

Kristensen, Malene M; Davidsen, Peter K; Vigelsø, Andreas; Hansen, Christina N; Jensen, Lars J; Jessen, Niels; Bruun, Jens M; Dela, Flemming; Helge, Jørn W

2017-03-01

Obesity is central in the development of insulin resistance. However, the underlying mechanisms still need elucidation. Dysregulated microRNAs (miRNAs; post-transcriptional regulators) in adipose tissue may present an important link. The miRNA expression in subcutaneous adipose tissue from 19 individuals with severe obesity (10 women and 9 men) before and after a 15-week weight loss intervention was studied using genome-wide microarray analysis. The microarray results were validated with RT-qPCR, and pathway enrichment analysis of in silico predicted targets was performed to elucidate the biological consequences of the miRNA dysregulation. Lastly, the messenger RNA (mRNA) and/or protein expression of multiple predicted targets as well as several proteins involved in lipolysis were investigated. The intervention led to upregulation of miR-29a-3p and miR-29a-5p and downregulation of miR-20b-5p. The mRNA and protein expression of predicted targets was not significantly affected by the intervention. However, negative correlations between miR-20b-5p and the protein levels of its predicted target, acyl-CoA synthetase long-chain family member 1, were observed. Several other miRNA-target relationships correlated negatively, indicating possible miRNA regulation, including miR-29a-3p and lipoprotein lipase mRNA levels. Proteins involved in lipolysis were not affected by the intervention. Weight loss influenced several miRNAs, some of which were negatively correlated with predicted targets. These dysregulated miRNAs may affect adipocytokine signaling and forkhead box protein O signaling. © 2017 The Obesity Society.

Gene expression analysis of the liver and skeletal muscle of psyllium-treated mice.

PubMed

Togawa, Naoyuki; Takahashi, Rumiko; Hirai, Shizuka; Fukushima, Tatsunobu; Egashira, Yukari

2013-02-14

Psyllium, a dietary fibre rich in soluble components, has both cholesterol- and TAG-lowering effects. Many studies have verified these actions using liver samples, whereas little information is available on the effects of psyllium treatment on other organs. The purpose of the present study was to evaluate the possible beneficial effects of psyllium. We investigated the gene expression profiles of both liver and skeletal muscle using DNA microarrays. C57BL/6J mice were fed a low-fat diet (LFD; 7 % fat), a high-fat diet (HFD; 40 % fat) or a HFD with psyllium (40 % fat+5 % psyllium; HFD+Psy) for 10 weeks. Body weights and food intake were measured weekly. After 10 weeks, the mice were killed and tissues were collected. Adipose tissues were weighed, and plasma total cholesterol and TAG blood glucose levels were measured. The expression levels of genes involved in glycolysis, gluconeogenesis, glucose transport and fatty acid metabolism were measured by DNA microarray in the liver and skeletal muscle. In the HFD+Psy group, plasma total cholesterol, TAG and blood glucose levels significantly decreased. There was a significant reduction in the relative weight of the epididymal and retroperitoneal fat tissue depots in mice fed the HFD+Psy. The expression levels of genes involved in fatty acid oxidation and lipid transport were significantly up-regulated in the skeletal muscle of the HFD+Psy group. This result suggests that psyllium stimulates lipid transport and fatty acid oxidation in the muscle. In conclusion, the present study demonstrates that psyllium can promote lipid consumption in the skeletal muscle; and this effect would create a slightly insufficient glucose state in the liver.

GeneAnalytics: An Integrative Gene Set Analysis Tool for Next Generation Sequencing, RNAseq and Microarray Data.

PubMed

Ben-Ari Fuchs, Shani; Lieder, Iris; Stelzer, Gil; Mazor, Yaron; Buzhor, Ella; Kaplan, Sergey; Bogoch, Yoel; Plaschkes, Inbar; Shitrit, Alina; Rappaport, Noa; Kohn, Asher; Edgar, Ron; Shenhav, Liraz; Safran, Marilyn; Lancet, Doron; Guan-Golan, Yaron; Warshawsky, David; Shtrichman, Ronit

2016-03-01

Postgenomics data are produced in large volumes by life sciences and clinical applications of novel omics diagnostics and therapeutics for precision medicine. To move from "data-to-knowledge-to-innovation," a crucial missing step in the current era is, however, our limited understanding of biological and clinical contexts associated with data. Prominent among the emerging remedies to this challenge are the gene set enrichment tools. This study reports on GeneAnalytics™ ( geneanalytics.genecards.org ), a comprehensive and easy-to-apply gene set analysis tool for rapid contextualization of expression patterns and functional signatures embedded in the postgenomics Big Data domains, such as Next Generation Sequencing (NGS), RNAseq, and microarray experiments. GeneAnalytics' differentiating features include in-depth evidence-based scoring algorithms, an intuitive user interface and proprietary unified data. GeneAnalytics employs the LifeMap Science's GeneCards suite, including the GeneCards®--the human gene database; the MalaCards-the human diseases database; and the PathCards--the biological pathways database. Expression-based analysis in GeneAnalytics relies on the LifeMap Discovery®--the embryonic development and stem cells database, which includes manually curated expression data for normal and diseased tissues, enabling advanced matching algorithm for gene-tissue association. This assists in evaluating differentiation protocols and discovering biomarkers for tissues and cells. Results are directly linked to gene, disease, or cell "cards" in the GeneCards suite. Future developments aim to enhance the GeneAnalytics algorithm as well as visualizations, employing varied graphical display items. Such attributes make GeneAnalytics a broadly applicable postgenomics data analyses and interpretation tool for translation of data to knowledge-based innovation in various Big Data fields such as precision medicine, ecogenomics, nutrigenomics, pharmacogenomics, vaccinomics, and others yet to emerge on the postgenomics horizon.

Human Endometriosis Tissue Microarray Reveals Site-specific Expression of Estrogen Receptors, Progesterone Receptor, and Ki67.

PubMed

Colón-Caraballo, Mariano; García, Miosotis; Mendoza, Adalberto; Flores, Idhaliz

2018-04-07

Most available therapies for endometriosis are hormone-based and generally broadly used without taking into consideration the ovarian hormone receptor expression status. This contrasts strikingly with the standard of care for other hormone-based conditions such as breast cancer. We therefore aimed to characterize the expression of ovarian steroid hormone receptors for estrogen alpha (ESR1), estrogen beta (ESR2), and progesterone (PGR) in different types of endometriotic lesions and eutopic endometrium from women with endometriosis and controls using a tissue microarray (TMA). Nuclear expression levels of the receptors were analyzed by tissue (ie, ectopic vs. eutopic endometrium) and cell type (ie, glands vs. stroma). Ovarian lesions showed the lowest expression of ESR1 and PGR, and the highest expression of ESR2, whereas the fallopian tube lesions showed high expression of the 3 receptors. Differences among endometria included lower expression of ESR1 and higher expression of ESR2 in stroma of proliferative endometrium from patients versus patients, and a trend towards loss of PGR nuclear positivity in proliferative endometrium from patients. The largest ESR2:ESR1 ratios were observed in ovarian lesions and secretory endometrium. The highest proportion of samples with >10% Ki67 positive nuclei was in glands of fallopian tube (54%) and extrapelvic lesions (75%); 60% of glands of secretory endometrium from patients had >10% Ki67 positivity compared with only 15% in controls. Our results provide a better understanding of endometriosis heterogeneity by revealing lesion type-specific differences and case-by-case variability in the expression of ovarian hormone receptors. This knowledge could potentially predict individual responses to hormone therapies, and set the basis for the application of personalized medicine approaches for women with endometriosis.

Identifying significant genetic regulatory networks in the prostate cancer from microarray data based on transcription factor analysis and conditional independency

PubMed Central

2009-01-01

Background Prostate cancer is a world wide leading cancer and it is characterized by its aggressive metastasis. According to the clinical heterogeneity, prostate cancer displays different stages and grades related to the aggressive metastasis disease. Although numerous studies used microarray analysis and traditional clustering method to identify the individual genes during the disease processes, the important gene regulations remain unclear. We present a computational method for inferring genetic regulatory networks from micorarray data automatically with transcription factor analysis and conditional independence testing to explore the potential significant gene regulatory networks that are correlated with cancer, tumor grade and stage in the prostate cancer. Results To deal with missing values in microarray data, we used a K-nearest-neighbors (KNN) algorithm to determine the precise expression values. We applied web services technology to wrap the bioinformatics toolkits and databases to automatically extract the promoter regions of DNA sequences and predicted the transcription factors that regulate the gene expressions. We adopt the microarray datasets consists of 62 primary tumors, 41 normal prostate tissues from Stanford Microarray Database (SMD) as a target dataset to evaluate our method. The predicted results showed that the possible biomarker genes related to cancer and denoted the androgen functions and processes may be in the development of the prostate cancer and promote the cell death in cell cycle. Our predicted results showed that sub-networks of genes SREBF1, STAT6 and PBX1 are strongly related to a high extent while ETS transcription factors ELK1, JUN and EGR2 are related to a low extent. Gene SLC22A3 may explain clinically the differentiation associated with the high grade cancer compared with low grade cancer. Enhancer of Zeste Homolg 2 (EZH2) regulated by RUNX1 and STAT3 is correlated to the pathological stage. Conclusions We provide a computational framework to reconstruct the genetic regulatory network from the microarray data using biological knowledge and constraint-based inferences. Our method is helpful in verifying possible interaction relations in gene regulatory networks and filtering out incorrect relations inferred by imperfect methods. We predicted not only individual gene related to cancer but also discovered significant gene regulation networks. Our method is also validated in several enriched published papers and databases and the significant gene regulatory networks perform critical biological functions and processes including cell adhesion molecules, androgen and estrogen metabolism, smooth muscle contraction, and GO-annotated processes. Those significant gene regulations and the critical concept of tumor progression are useful to understand cancer biology and disease treatment. PMID:20025723

The Use of P63 Immunohistochemistry for the Identification of Squamous Cell Carcinoma of the Lung

PubMed Central

Conde, Esther; Angulo, Bárbara; Redondo, Pilar; Toldos, Oscar; García-García, Elena; Suárez-Gauthier, Ana; Rubio-Viqueira, Belén; Marrón, Carmen; García-Luján, Ricardo; Sánchez-Céspedes, Montse; López-Encuentra, Angel; Paz-Ares, Luis; López-Ríos, Fernando

2010-01-01

Introduction While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of “carcinoma NOS (not otherwise specified)” in a prospective series of small tumor samples. Methods With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC. Results The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p<0.0001); (2) half of the 20 (50%) LCCs were positive for P63 and were reclassified as SCCs; and (3) all P63 positive cases (34%) were diagnosed as SCCs. Conclusions P63 IHC is useful for the identification of lung SCCs. PMID:20808915

Transcriptomic Analysis Using Olive Varieties and Breeding Progenies Identifies Candidate Genes Involved in Plant Architecture.

PubMed

González-Plaza, Juan J; Ortiz-Martín, Inmaculada; Muñoz-Mérida, Antonio; García-López, Carmen; Sánchez-Sevilla, José F; Luque, Francisco; Trelles, Oswaldo; Bejarano, Eduardo R; De La Rosa, Raúl; Valpuesta, Victoriano; Beuzón, Carmen R

2016-01-01

Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Little is known about how plant architecture is genetically determined in olive, were most of the existing varieties are traditional with an architecture poorly suited for modern growing and harvesting systems. In the present study, we have carried out microarray analysis of meristematic tissue to compare expression profiles of olive varieties displaying differences in architecture, as well as seedlings from their cross pooled on the basis of their sharing architecture-related phenotypes. The microarray used, previously developed by our group has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. Varieties with distinct architecture phenotypes and individuals from segregating progenies displaying opposite architecture features were used to link phenotype to expression. Here, we identify 2252 differentially expressed genes (DEGs) associated to differences in plant architecture. Microarray results were validated by quantitative RT-PCR carried out on genes with functional annotation likely related to plant architecture. Twelve of these genes were further analyzed in individual seedlings of the corresponding pool. We also examined Arabidopsis mutants in putative orthologs of these targeted candidate genes, finding altered architecture for most of them. This supports a functional conservation between species and potential biological relevance of the candidate genes identified. This study is the first to identify genes associated to plant architecture in olive, and the results obtained could be of great help in future programs aimed at selecting phenotypes adapted to modern cultivation practices in this species.

Toxicity evaluation of glyphosate agrochemical components using Japanese medaka (Oryzias latipes) and DNA microarray gene expression analysis.

PubMed

Uchida, Masaya; Takumi, Shota; Tachikawa, Keiko; Yamauchi, Ryoko; Goto, Yoshiyuki; Matsusaki, Hiromi; Nakamura, Hiroshi; Kagami, Yoshihiro; Kusano, Teruhiko; Arizono, Koji

2012-01-01

Using glyphosate agrochemical components, we investigated their acute toxicity to juvenile Japanese medaka (Oryzias latipes) as well as their toxic impact at gene expression level on the liver tissues of adult medaka using DNA microarray. In our acute toxicity test, juvenile medaka were exposed for 96 hr to each of the following glyphosate agrochemical components: 10~160 mg/l of glyphosate, 1.25~20 mg/l of fatty acid alkanolamide surfactant (DA), and 12~416 mg/l of a fully formulated glyphosate herbicide. As a result, LC(50) values of glyphosate, DA, and the glyphosate herbicide were > 160 mg/l, 8.5 mg/l, and 76.8 mg/l, respectively. On the other hand, adult male medaka fish were exposed to each of the glyphosate agrochemical components for 48 hr at the following concentrations: 16 mg/l of glyphosate, 0.5 mg/l of DA, and 16 mg/l-glyphosate/0.5 mg/l-DA mixture. Interestingly, DNA microarray analysis revealed that there were no significant gene expression changes in the medaka liver after exposure to glyphosate. Nevertheless, 78 and 138 genes were significantly induced by DA and the glyphosate/DA mixture, respectively. Furthermore, we identified five common genes that were affected by DA and glyphosate/DA mixture. These results suggested that glyphosate itself possessed very low toxicity as previously reported by some researchers at least to the small laboratory fish, and the major toxicity of the glyphosate agrochemical resided mainly in DA and perhaps in unintentionally generated byproduct(s) of glyphosate-DA mixture.

Joint mapping of genes and conditions via multidimensional unfolding analysis

PubMed Central

Van Deun, Katrijn; Marchal, Kathleen; Heiser, Willem J; Engelen, Kristof; Van Mechelen, Iven

2007-01-01

Background Microarray compendia profile the expression of genes in a number of experimental conditions. Such data compendia are useful not only to group genes and conditions based on their similarity in overall expression over profiles but also to gain information on more subtle relations between genes and conditions. Getting a clear visual overview of all these patterns in a single easy-to-grasp representation is a useful preliminary analysis step: We propose to use for this purpose an advanced exploratory method, called multidimensional unfolding. Results We present a novel algorithm for multidimensional unfolding that overcomes both general problems and problems that are specific for the analysis of gene expression data sets. Applying the algorithm to two publicly available microarray compendia illustrates its power as a tool for exploratory data analysis: The unfolding analysis of a first data set resulted in a two-dimensional representation which clearly reveals temporal regulation patterns for the genes and a meaningful structure for the time points, while the analysis of a second data set showed the algorithm's ability to go beyond a mere identification of those genes that discriminate between different patient or tissue types. Conclusion Multidimensional unfolding offers a useful tool for preliminary explorations of microarray data: By relying on an easy-to-grasp low-dimensional geometric framework, relations among genes, among conditions and between genes and conditions are simultaneously represented in an accessible way which may reveal interesting patterns in the data. An additional advantage of the method is that it can be applied to the raw data without necessitating the choice of suitable genewise transformations of the data. PMID:17550582

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums.

PubMed

Shakoor, Nadia; Nair, Ramesh; Crasta, Oswald; Morris, Geoffrey; Feltus, Alex; Kresovich, Stephen

2014-01-23

Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e.g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.

PTEN loss and chromosome 8 alterations in Gleason grade 3 prostate cancer cores predicts the presence of un-sampled grade 4 tumor: implications for active surveillance.

PubMed

Trock, Bruce J; Fedor, Helen; Gurel, Bora; Jenkins, Robert B; Knudsen, B S; Fine, Samson W; Said, Jonathan W; Carter, H Ballentine; Lotan, Tamara L; De Marzo, Angelo M

2016-07-01

Men who enter active surveillance because their biopsy exhibits only Gleason grade 3 (G3) frequently have higher grade tumor missed by biopsy. Thus, biomarkers are needed that, when measured on G3 tissue, can predict the presence of higher grade tumor in the whole prostate. We evaluated whether PTEN loss, chromosome 8q gain (MYC) and/or 8p loss (LPL) measured only on G3 cores is associated with un-sampled G4 tumor. A tissue microarray was constructed of prostatectomy tissue from patients whose prostates exhibited only Gleason score 3+3, only 3+4 or only 4+3 tumor (n=50 per group). Cores sampled only from areas of G3 were evaluated for PTEN loss by immunohistochemistry, and PTEN deletion, LPL/8p loss and MYC/8q gain by fluorescence in situ hybridization. Biomarker results were compared between Gleason score 6 vs 7 tumors using conditional logistic regression. PTEN protein loss, odds ratio=4.99, P=0.033; MYC/8q gain, odds ratio=5.36, P=0.010; and LPL/8p loss, odds ratio=3.96, P=0.003 were significantly more common in G3 cores derived from Gleason 7 vs Gleason 6 tumors. PTEN gene deletion was not statistically significant. Associations were stronger comparing Gleason 4+3 vs 6 than for Gleason 3+4 vs 6. MYC/8q gain, LPL/8p loss and PTEN protein loss measured in G3 tissue microarray cores strongly differentiate whether the core comes from a Gleason 6 or Gleason 7 tumor. If validated to predict upgrading from G3 biopsy to prostatectomy these biomarkers could reduce the likelihood of enrolling high-risk men and facilitate safe patient selection for active surveillance.

MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4

PubMed Central

Zhang, Dongyuan; Sun, Hongcheng; Yu, Fudong; Yue, Ben; Wang, Jingtao; Zhang, Meng; Yu, Yang; Liu, Xisheng; Sun, Xiaofeng; Zhou, Zongguang; Qin, Xuebin; Zhang, Xin; Yan, Dongwang; Wen, Yugang; Peng, Zhihai

2016-01-01

Background Tumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known. Results miR-20a-5p negatively regulated Smad4 by directly targeting its 3′UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo, but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients’ clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients. Methods Five miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo. The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan–Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data. Conclusions miR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer. PMID:27286257

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6.

PubMed

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-07-15

To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection.

Deregulation of energetic metabolism in the clear cell renal cell carcinoma: A multiple pathway analysis based on microarray profiling.

PubMed

Soltysova, Andrea; Breza, Jan; Takacova, Martina; Feruszova, Jana; Hudecova, Sona; Novotna, Barbora; Rozborilova, Eva; Pastorekova, Silvia; Kadasi, Ludevit; Krizanova, Olga

2015-07-01

Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. In order to better understand the biology of ccRCC, we accomplished the gene profiling of fresh tissue specimens from 11 patients with the renal tumors (9 ccRCCs, 1 oncocytoma and 1 renal B-lymphoma), in which the tumor-related data were compared to the paired healthy kidney tissues from the same patients. All ccRCCs exhibited a considerably elevated transcription of the gene coding for carbonic anhydrase IX (CAIX). Moreover, the ccRCC tumors consistently displayed increased expression of genes encoding the glycolytic pathway enzymes, e.g. hexokinase II (HK2) and lactate dehydrogenase A (LDHA) and a decreased expression of genes for the mitochondrial electron transport chain components, indicating an overall reprogramming of the energetic metabolism in this tumor type. This appears to be accompanied by altered expression of the genes of the pH regulating machinery, including ion and lactate transporters. Immunohistochemical staining of tumor tissue sections confirmed the increased expression of CAIX, HK2 and LDHA in ccRCC, validating the microarray data and supporting their potential as the energetic metabolism-related biomarkers of the ccRCC.

Expression Profile of Long Noncoding RNAs in Human Earlobe Keloids: A Microarray Analysis

PubMed Central

Guo, Liang; Xu, Kai; Yan, Hongbo; Feng, Haifeng

2016-01-01

Background. Long noncoding RNAs (lncRNAs) play key roles in a wide range of biological processes and their deregulation results in human disease, including keloids. Earlobe keloid is a type of pathological skin scar, and the molecular pathogenesis of this disease remains largely unknown. Methods. In this study, microarray analysis was used to determine the expression profiles of lncRNAs and mRNAs between 3 pairs of earlobe keloid and normal specimens. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify the main functions of the differentially expressed genes and earlobe keloid-related pathways. Results. A total of 2068 lncRNAs and 1511 mRNAs were differentially expressed between earlobe keloid and normal tissues. Among them, 1290 lncRNAs and 1092 mRNAs were upregulated, and 778 lncRNAs and 419 mRNAs were downregulated. Pathway analysis revealed that 24 pathways were correlated to the upregulated transcripts, while 11 pathways were associated with the downregulated transcripts. Conclusion. We characterized the expression profiles of lncRNA and mRNA in earlobe keloids and suggest that lncRNAs may serve as diagnostic biomarkers for the therapy of earlobe keloid. PMID:28101509

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

PubMed

Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

2017-04-01

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in the formation and development of hepatocellular carcinoma and that peroxisome proliferator-activated receptor gamma coactivator-1 alpha may be a potential therapeutic target for hepatocellular carcinoma.

Comprehensive adipocytic and neurogenic tissue microarray analysis of NY-ESO-1 expression - a promising immunotherapy target in malignant peripheral nerve sheath tumor and liposarcoma

PubMed Central

Shurell, Elizabeth; Vergara-Lluri, Maria E.; Li, Yunfeng; Crompton, Joseph G.; Singh, Arun; Bernthal, Nicholas; Wu, Hong; Eilber, Fritz C.; Dry, Sarah M.

2016-01-01

Background Immunotherapy targeting cancer-testis antigen NY-ESO-1 shows promise for tumors with poor response to chemoradiation. Malignant peripheral nerve sheath tumors (MPNSTs) and liposarcomas (LPS) are chemoresistant and have few effective treatment options. Materials Methods Using a comprehensive tissue microarray (TMA) of both benign and malignant tumors in primary, recurrent, and metastatic samples, we examined NY-ESO-1 expression in peripheral nerve sheath tumor (PNST) and adipocytic tumors. The PNST TMA included 42 MPNSTs (spontaneous n = 26, NF1-associated n = 16), 35 neurofibromas (spontaneous n = 22, NF-1 associated n = 13), 11 schwannomas, and 18 normal nerves. The LPS TMA included 48 well-differentiated/dedifferentiated (WD/DD) LPS, 13 myxoid/round cell LPS, 3 pleomorphic LPS, 8 lipomas, 1 myelolipoma, and 3 normal adipocytic tissue samples. Stained in triplicate, NY-ESO-1 intensity and density were scored. Results NY-ESO-1 expression was exclusive to malignant tumors. 100% of myxoid/round cell LPS demonstrated NY-ESO-1 expression, while only 6% of WD/DD LPS showed protein expression, one of which was WD LPS. Of MPNST, 4/26 (15%) spontaneous and 2/16 (12%) NF1-associated MPNSTs demonstrated NY-ESO-1 expression. Strong NY-ESO-1 expression was observed in myxoid/round cell and dedifferentiated LPS, and MPNST in primary, neoadjuvant, and metastatic settings. Conclusions We found higher prevalence of NY-ESO-1 expression in MPNSTs than previously reported, highlighting a subset of MPNST patients who may benefit from immunotherapy. This study expands our understanding of NY-ESO-1 in WD/DD LPS and is the first demonstration of staining in a WD LPS and metastatic/recurrent myxoid/round cell LPS. These results suggest immunotherapy targeting NY-ESO-1 may benefit patients with aggressive tumors resistant to conventional therapy. PMID:27655679

Larger core size has superior technical and analytical accuracy in bladder tissue microarray.

PubMed

Eskaros, Adel Rh; Egloff, Shanna A Arnold; Boyd, Kelli L; Richardson, Joyce E; Hyndman, M Eric; Zijlstra, Andries

2017-03-01

The construction of tissue microarrays (TMAs) with cores from a large number of paraffin-embedded tissues (donors) into a single paraffin block (recipient) is an effective method of analyzing samples from many patient specimens simultaneously. For the TMA to be successful, the cores within it must capture the correct histologic areas from the donor blocks (technical accuracy) and maintain concordance with the tissue of origin (analytical accuracy). This can be particularly challenging for tissues with small histological features such as small islands of carcinoma in situ (CIS), thin layers of normal urothelial lining of the bladder, or cancers that exhibit intratumor heterogeneity. In an effort to create a comprehensive TMA of a bladder cancer patient cohort that accurately represents the tumor heterogeneity and captures the small features of normal and CIS, we determined how core size (0.6 vs 1.0 mm) impacted the technical and analytical accuracy of the TMA. The larger 1.0 mm core exhibited better technical accuracy for all tissue types at 80.9% (normal), 94.2% (tumor), and 71.4% (CIS) compared with 58.6%, 85.9%, and 63.8% for 0.6 mm cores. Although the 1.0 mm core provided better tissue capture, increasing the number of replicates from two to three allowed with the 0.6 mm core compensated for this reduced technical accuracy. However, quantitative image analysis of proliferation using both Ki67+ immunofluorescence counts and manual mitotic counts demonstrated that the 1.0 mm core size also exhibited significantly greater analytical accuracy (P=0.004 and 0.035, respectively, r 2 =0.979 and 0.669, respectively). Ultimately, our findings demonstrate that capturing two or more 1.0 mm cores for TMA construction provides superior technical and analytical accuracy over the smaller 0.6 mm cores, especially for tissues harboring small histological features or substantial heterogeneity.

Genotyping microarray: Mutation screening in Spanish families with autosomal dominant retinitis pigmentosa

PubMed Central

García-Hoyos, María; Cortón, Marta; Ávila-Fernández, Almudena; Riveiro-Álvarez, Rosa; Giménez, Ascensión; Hernan, Inma; Carballo, Miguel; Ayuso, Carmen

2012-01-01

Purpose Presently, 22 genes have been described in association with autosomal dominant retinitis pigmentosa (adRP); however, they explain only 50% of all cases, making genetic diagnosis of this disease difficult and costly. The aim of this study was to evaluate a specific genotyping microarray for its application to the molecular diagnosis of adRP in Spanish patients. Methods We analyzed 139 unrelated Spanish families with adRP. Samples were studied by using a genotyping microarray (adRP). All mutations found were further confirmed with automatic sequencing. Rhodopsin (RHO) sequencing was performed in all negative samples for the genotyping microarray. Results The adRP genotyping microarray detected the mutation associated with the disease in 20 of the 139 families with adRP. As in other populations, RHO was found to be the most frequently mutated gene in these families (7.9% of the microarray genotyped families). The rate of false positives (microarray results not confirmed with sequencing) and false negatives (mutations in RHO detected with sequencing but not with the genotyping microarray) were established, and high levels of analytical sensitivity (95%) and specificity (100%) were found. Diagnostic accuracy was 15.1%. Conclusions The adRP genotyping microarray is a quick, cost-efficient first step in the molecular diagnosis of Spanish patients with adRP. PMID:22736939

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray

PubMed Central

2009-01-01

Background It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Results Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. Conclusion The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction. PMID:19825185

A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

PubMed Central

Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

2016-01-01

A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids released via lipolysis of white adipose tissue. PMID:27187182

In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics

PubMed Central

Grundberg, Ida; Kiflemariam, Sara; Mignardi, Marco; Imgenberg-Kreuz, Juliana; Edlund, Karolina; Micke, Patrick; Sundström, Magnus; Sjöblom, Tobias

2013-01-01

Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy. PMID:24280411

Immunohistochemical characterization of neoplastic cells of breast origin.

PubMed

Noriega, Mariadelasmercedes; Paesani, Fernando; Perazzo, Florencia; Lago, Néstor; Krupitzki, Hugo; Nieto, Silvana; Garcia, Alejandro; Avagnina, Alejandra; Elsner, Boris; Denninghoff, Valeria Cecilia

2012-06-22

After skin cancer, breast cancer is the most common malignancy in women. Tumors of unknown origin account for 5-15% of malignant neoplasms, with 1.5% being breast cancer. An immunohistochemical panel with conventional and newer markers, such as mammaglobin, was selected for the detection of neoplastic cells of breast origin. The specific objectives are: 1) to determine the sensitivity and specificity of the panel, with a special emphasis on the inclusion of the mammaglobin marker, and 2) to compare immunohistochemistry performed on whole tissue sections and on tissue micro-array. Twenty-nine metastatic breast tumors were included and assumed as tumors of unknown origin. Other 48 biopsies of diverse tissues were selected and assumed as negative controls. Tissue Micro-Array was performed. Immunohistochemistry for mammaglobin, gross cystic disease fluid protein-15, estrogen receptor, progesterone receptor and cytokeratin 7 was done. Mammaglobin positive staining was observed in 10/29 cases, in 13/29 cases for gross cystic disease fluid protein-15, in 20/29 cases for estrogen receptor, in 9/29 cases for progesterone receptor, and in 25/29 cases for cytokeratin 7. Among the negative controls, mammaglobin was positive in 2/48, and gross cystic disease fluid protein-15 in 4/48. The inclusion of MAG antibody in the immunohistochemical panel for the detection of tumors of unknown origin contributed to the detection of metastasis of breast cancer. The diagnostic strategy with the highest positive predictive value (88%) included hormone receptors and mammaglobin in serial manner.

Inhibition of myostatin protects against diet-induced obesity by enhancing fatty acid oxidation and promoting a brown adipose phenotype in mice.

PubMed

Zhang, C; McFarlane, C; Lokireddy, S; Masuda, S; Ge, X; Gluckman, P D; Sharma, M; Kambadur, R

2012-01-01

Although myostatin-null (Mstn (-/-)) mice fail to accumulate fat in adipose tissue when fed a high-fat diet (HFD), little is known about the molecular mechanism(s) behind this phenomenon. We therefore sought to identify the signalling pathways through which myostatin regulates accumulation and/or utilisation of fat. Wild-type, Mstn (-/-) and wild-type mice treated with soluble activin type IIB receptor (sActRIIB) were fed a control chow diet or an HFD for 12 weeks. Changes in gene expression were measured by microarray and quantitative PCR. Histological changes in white adipose tissue were assessed together with peripheral tissue fatty acid oxidation and changes in circulating hormones following HFD feeding. Our results demonstrate that inactivation of myostatin results in reduced fat accumulation in mice on an HFD. Molecular analysis revealed that metabolic benefits, due to lack of myostatin, are mediated through at least two independent mechanisms. First, lack of myostatin increased fatty acid oxidation in peripheral tissues through induction of enzymes involved in lipolysis and in fatty acid oxidation in mitochondria. Second, inactivation of myostatin also enhanced brown adipose formation in white adipose tissue of Mstn (-/-) mice. Consistent with the above, treatment of HFD-fed wild-type mice with the myostatin antagonist, sActRIIB, reduced the obesity phenotype. We conclude that absence of myostatin results in enhanced peripheral tissue fatty acid oxidation and increased thermogenesis, culminating in increased fat utilisation and reduced adipose tissue mass. Taken together, our data suggest that anti-myostatin therapeutics could be beneficial in alleviating obesity.

Gene expression profiles help identify the tissue of origin for metastatic brain cancers.

PubMed

Wu, Alan H B; Drees, Julia C; Wang, Hangpin; VandenBerg, Scott R; Lal, Anita; Henner, William D; Pillai, Raji

2010-04-26

Metastatic brain cancers are the most common intracranial tumor and occur in about 15% of all cancer patients. In up to 10% of these patients, the primary tumor tissue remains unknown, even after a time consuming and costly workup. The Pathwork Tissue of Origin Test (Pathwork Diagnostics, Redwood City, CA, USA) is a gene expression test to aid in the diagnosis of metastatic, poorly differentiated and undifferentiated tumors. It measures the expression pattern of 1,550 genes in these tumors and compares it to the expression pattern of a panel of 15 known tumor types. The purpose of this study was to evaluate the performance of the Tissue of Origin Test in the diagnosis of primary sites for metastatic brain cancer patients. Fifteen fresh-frozen metastatic brain tumor specimens of known origins met specimen requirements. These specimens were entered into the study and processed using the Tissue of Origin Test. Results were compared to the known primary site and the agreement between the two results was assessed. Fourteen of the fifteen specimens produced microarray data files that passed all quality metrics. One originated from a tissue type that was off-panel. Among the remaining 13 cases, the Tissue of Origin Test accurately predicted the available diagnosis in 12/13 (92.3%) cases. This study demonstrates the accuracy of the Tissue of Origin Test when applied to predict the tissue of origin of metastatic brain tumors. This test could be a very useful tool for pathologists as they classify metastatic brain cancers.

Microarray-integrated optoelectrofluidic immunoassay system

PubMed Central

Han, Dongsik

2016-01-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection. PMID:27190571

Microarray-integrated optoelectrofluidic immunoassay system.

PubMed

Han, Dongsik; Park, Je-Kyun

2016-05-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

West Nile virus infection in killer whale, Texas, USA, 2007.

PubMed

St Leger, Judy; Wu, Guang; Anderson, Mark; Dalton, Les; Nilson, Erika; Wang, David

2011-08-01

In 2007, nonsuppurative encephalitis was identified in a killer whale at a Texas, USA, marine park. Panviral DNA microarray of brain tissue suggested West Nile virus (WNV); WNV was confirmed by reverse transcription PCR and sequencing. Immunohistochemistry demonstrated WNV antigen within neurons. WNV should be considered in cases of encephalitis in cetaceans.

Differences in X-chromosome transcriptional activity and cholesterol metabolism between placentae from swine breeds from asian and western origins

USDA-ARS?s Scientific Manuscript database

To gain insight into placental physiology differences between the Chinese Meishan and white composite (WC) swine breeds, short-oligonucleotide microarray gene expression profiles of gestational Day 25, 45, 65, 85, and 105 placental tissues were compared. Differential expression was determined by a ...

Endoglin (CD105) expression on microvessel endothelial cells in juvenile nasopharyngeal angiofibroma: tissue microarray analysis and association with prognostic significance.

PubMed

Wang, Jing-Jing; Sun, Xi-Cai; Hu, Li; Liu, Zhuo-Fu; Yu, Hua-Peng; Li, Han; Wang, Shu-Yi; Wang, De-Hui

2013-12-01

The purpose of this study was to examine endoglin (CD105) expression on microvessel endothelial cells (ECs) in juvenile nasopharyngeal angiofibroma (JNA) and its relationship with recurrence. Immunohistochemistry was performed to detect CD105 expression in a tissue microarray from 70 patients with JNA. Correlation between CD105 expression on microvessel ECs and clinicopathological features, as well as tumor recurrence, were analyzed. Immunohistochemistry revealed CD105 expression on ECs but not in stroma of patients with JNA. Chi-square analysis indicated CD105-based microvessel density (MVD) was correlated with JNA recurrence (p = .013). Univariate and multivariate analyses determined that MVD was a significant predictor of time to recurrence (p = .009). The CD105-based MVD was better for predicting disease recurrence (AUROC: 0.673; p = .036) than other clinicopathological features. MVD is a useful predictor for poor prognosis of patients with JNA after curative resection. Angiogenesis, which may play an important role in the occurrence and development of JNA, is therefore a potential therapeutic target for JNA. Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.

Circular RNA hsa_circ_0008344 regulates glioblastoma cell proliferation, migration, invasion, and apoptosis.

PubMed

Zhou, Jinxu; Wang, Hongxiang; Chu, Junsheng; Huang, Qilin; Li, Guangxu; Yan, Yong; Xu, Tao; Chen, Juxiang; Wang, Yuhai

2018-04-24

Recent studies have found circular RNAs (circRNAs) involved in the biological process of cancers. However, little is known about their functional roles in glioblastoma. Human circRNA microarray analysis was performed to screen the expression profile of circRNAs in IDH1 wild-type glioblastoma tissue. The expression of hsa_circ_0008344 in glioblastoma and normal brain samples was quantified by qRT-PCR. Functional experiments were performed to investigate the biological functions of hsa_circ_0008344, including MTT assay, colony formation assay, transwell assay, and cell apoptosis assay. CircRNA microarray revealed a total of 417 abnormally expressed circRNAs (>1.5-fold, P < .05) in glioblastoma tissue compared with the adjacent normal brain. Hsa_circ_0008344, among the top differentially expressed circRNAs, was significantly upregulated in IDH1 wild-type glioblastoma. Further in vitro studies showed that knockdown of hsa_circ_0008344 suppressed glioblastoma cell proliferation, colony formation, migration, and invasion, but increased cell apoptotic rate. Hsa_circ_0008344 is upregulated in glioblastoma and may contribute to the progression of this malignancy. © 2018 Wiley Periodicals, Inc.

Characterisation of the cancer-associated glucocorticoid system: key role of 11β-hydroxysteroid dehydrogenase type 2.

PubMed

Cirillo, Nicola; Morgan, David J; Pedicillo, Maria Carmela; Celentano, Antonio; Lo Muzio, Lorenzo; McCullough, Michael J; Prime, Stephen S

2017-09-26

Recent studies have shown that production of cortisol not only takes place in several non-adrenal peripheral tissues such as epithelial cells but, also, the local inter-conversion between cortisone and cortisol is regulated by the 11β-hydroxysteroid dehydrogenases (11β-HSDs). However, little is known about the activity of this non-adrenal glucocorticoid system in cancers. The presence of a functioning glucocorticoid system was assessed in human skin squamous cell carcinoma (SCC) and melanoma and further, in 16 epithelial cell lines from 8 different tissue types using ELISA, western blotting and immunofluorescence. 11β-HSD2 was inhibited both pharmacologically and by siRNA technology. Naïve CD8 + T cells were used to test the paracrine effects of cancer-derived cortisol on the immune system in vitro. Functional assays included cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical data of 11β-HSD expression were generated using tissue microarrays of 40 cases of human SCCs as well as a database featuring 315 cancer cases from 15 different tissues. We show that cortisol production is a common feature of malignant cells and has paracrine functions. Cortisol production correlated with the magnitude of glucocorticoid receptor (GR)-dependent inhibition of tumour-specific CD8 + T cells in vitro. 11β-HSDs were detectable in human skin SCCs and melanoma. Analyses of publicly available protein expression data of 11β-HSDs demonstrated that 11β-HSD1 and -HSD2 were dysregulated in the majority (73%) of malignancies. Pharmacological manipulation of 11β-HSD2 activity by 18β-glycyrrhetinic acid (GA) and silencing by specific siRNAs modulated the bioavailability of cortisol. Cortisol also acted in an autocrine manner and promoted cell invasion in vitro and cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical analyses using tissue microarrays showed that expression of 11β-HSD2 was significantly reduced in human SCCs of the skin. The results demonstrate evidence of a cancer-associated glucocorticoid system and show for the first time, the functional significance of cancer-derived cortisol in tumour progression.

Expression of truncated Int6/eIF3e in mammary alveolar epithelium leads to persistent hyperplasia and tumorigenesis

PubMed Central

Mack, David L; Boulanger, Corinne A; Callahan, Robert; Smith, Gilbert H

2007-01-01

Introduction Int6 has been shown to be an interactive participant with the protein translation initiation complex eIF3, the COP9 signalosome and the regulatory lid of the 26S proteasome. Insertion of mouse mammary tumor virus into the Int6 locus creates a C-terminally truncated form of the protein. Expression of the truncated form of Int6 (Int6sh) in stably transfected human and mouse mammary epithelial cell lines leads to cellular transformation. In addition, decreased expression of Int6/eIF3e is observed in approximately one third of all human breast carcinomas. Methods To validate that Int6sh has transforming activity in vivo, a transgenic mouse model was designed using the whey acidic protein (Wap) promoter to target expression of truncated Int6 to differentiating alveolar epithelial cells in the mammary gland. Microarray analyses were performed on normal, premalignant and malignant WapInt6sh expressing tissues. Results Mammary tumors developed in 42% of WapInt6sh heterozygous parous females at an average age of 18 months. In WapInt6sh mice, the contralateral mammary glands from both tumorous and non-tumorous tissues contained widespread focal alveolar hyperplasia. Only 4% of WapInt6sh non-breeding females developed tumors by 2 years of age. The Wap promoter is active only during estrus in the mammary tissue of cycling non-pregnant mice. Microarray analyses of mammary tissues demonstrated that Int6sh expression in the alveolar tissue altered the mammary transcriptome in a specific manner that was detectable even in the first pregnancy. This Int6sh-specific transcriptome pattern subsequently persisted in both the Int6sh-expressing alveolar hyperplasia and mammary tumors. These observations are consistent with the conclusion that WapInt6sh-expressing alveolar cells survive involution following the cessation of lactation, and subsequently give rise to the mammary tumors that arise in aging multiparous females. Conclusion These observations provide direct in vivo evidence that mammary-specific expression of the Int6sh truncation leads to persistence of alveolar hyperplasia with the accompanying increased predisposition to mammary tumorigenesis. PMID:17626637

ROKU: a novel method for identification of tissue-specific genes.

PubMed

Kadota, Koji; Ye, Jiazhen; Nakai, Yuji; Terada, Tohru; Shimizu, Kentaro

2006-06-12

One of the important goals of microarray research is the identification of genes whose expression is considerably higher or lower in some tissues than in others. We would like to have ways of identifying such tissue-specific genes. We describe a method, ROKU, which selects tissue-specific patterns from gene expression data for many tissues and thousands of genes. ROKU ranks genes according to their overall tissue specificity using Shannon entropy and detects tissues specific to each gene if any exist using an outlier detection method. We evaluated the capacity for the detection of various specific expression patterns using synthetic and real data. We observed that ROKU was superior to a conventional entropy-based method in its ability to rank genes according to overall tissue specificity and to detect genes whose expression pattern are specific only to objective tissues. ROKU is useful for the detection of various tissue-specific expression patterns. The framework is also directly applicable to the selection of diagnostic markers for molecular classification of multiple classes.

Molecular pathway activation in cancer and tissue following space radiation exposure

NASA Astrophysics Data System (ADS)

Kovyrshina, Tatiana A.

Space radiation exposure is an important safety concern for astronauts, especially since one of the risks is carcinogenesis. This thesis explores the link between lung, colorectal, and breast cancer and iron particles and gamma radiation on a molecular level. We obtained DNA microarrays for each condition from the Gene Expression Omnibus (GEO), a public functional genomics data repository, cleaned up the data, and analysed overexpression and underexpression of pathway analysis. Our results show that pathways which participate in DNA replication appear to be overexpressed in cancer cells and cells exposed to ionizing radiation.

A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines.

PubMed

de Lima, Júlio César; Füller, Thanise Nogueira; de Costa, Fernanda; Rodrigues-Corrêa, Kelly C S; Fett-Neto, Arthur G

2016-01-01

RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

Profile of differentially expressed miRNAs in high-grade serous carcinoma and clear cell ovarian carcinoma, and the expression of miR-510 in ovarian carcinoma.

PubMed

Zhang, Xinchen; Guo, Gordon; Wang, Guang; Zhao, Jinyao; Wang, Bo; Yu, Xiaotang; Ding, Yanfang

2015-12-01

Improved insight into the molecular and genetic profile of different types of epithelial ovarian cancer (EOC) is required for understanding the carcinogenesis of EOC and may potentially be exploited by future targeted therapies. The aim of the present study was to identify a unique microRNA (miRNA) patterns and key miRNAs, which may assist in predicting progression and prognosis in high‑grade serous carcinoma (HGSC) and clear cell carcinoma (CCC). To identify unique miRNA patterns associated with HGSC and CCC, a miRNA microarray was performed using Chinese tumor bank specimens of patients with HGSC or CCC in a retrospective analysis. The expression levels of four deregulated miRNAs were further validated using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) in an external cohort of 42 cases of HGSC and 36 cases of CCC. Kaplan‑Meier analysis was performed to analyze the correlation between the expression levels of the four miRNAs and patient prognosis. Among these validated miRNAs, miR‑510 was further examined in another cohort of normal ovarian tissues, as well as the HGSC, low‑grade serous carcinoma (LGSC) and CCC specimens using RT‑qPCR and in situ hybridization. The results revealed that, of the 768 miRNAs analyzed in the microarray, 33 and 50 miRNAs were significantly upregulated and downregulated, respectively, with at least a 2‑fold difference in HGSC, compared with CCC. The quantitative analysis demonstrated that miR‑510 and miR‑129‑3p were significantly downregulated, and that miR‑483‑5p and miR‑miR‑449a were significantly upregulated in CCC, compared with HGSC (P<0.05), which was consistent with the microarray results. Kaplan‑Meier analysis revealed low expression levels of miR‑510 and low expression levels of miR‑129‑3p, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymphatic metastasis and that HGSC was significantly associated with the poorer overall survival rates (P<0.05). The expression of miR‑510 was significantly higher in the LGSC and CCC tissues, compared with the HGSC and normal ovarian tissues. The results of the present study suggested that different subtypes of EOC have specific miRNA signatures, and that miR‑510 may be involved differently in HGSC and CCC. Thus, miR‑510 and miR‑129‑3p may be considered as potential novel candidate clinical biomarkers for predicting the outcome of EOC.

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome.

PubMed

Morine, Melissa J; McMonagle, Jolene; Toomey, Sinead; Reynolds, Clare M; Moloney, Aidan P; Gormley, Isobel C; Gaora, Peadar O; Roche, Helen M

2010-10-07

Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways--selenoamino acid metabolism and steroid biosynthesis--illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease.

Extraction and labeling methods for microarrays using small amounts of plant tissue.

PubMed

Stimpson, Alexander J; Pereira, Rhea S; Kiss, John Z; Correll, Melanie J

2009-03-01

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).

Gene expression microarray analysis encompassing metamorphosis and the onset of calcification in the scleractinian coral Montastraea faveolata.

PubMed

Reyes-Bermudez, Alejandro; Desalvo, Michael K; Voolstra, Christian R; Sunagawa, Shinichi; Szmant, Alina M; Iglesias-Prieto, Roberto; Medina, Mónica

2009-01-01

Similar to many marine invertebrates, scleractinian corals experience a dramatic morphological transformation, as well as a habitat switch, upon settlement and metamorphosis. At this time, planula larvae transform from non-calcifying, demersal, motile organisms into sessile, calcifying, benthic juvenile polyps. We performed gene expression microarray analyses between planulae, aposymbiotic primary polyps, and symbiotic adult tissue to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification in the Robust/Short clade scleractinian coral Montastraea faveolata. Among the annotated genes, the most abundant upregulated transcripts in the planula stage are involved in protein synthesis, chromatin assembly and mitochondrial metabolism; the polyp stage, morphogenesis, protein catabolism and organic matrix synthesis; and the adult stage, sexual reproduction, stress response and symbiosis. We also present evidence showing that the planula and adult transcriptomes are more similar to each other than to the polyp transcriptome. Our results also point to a large number of uncharacterized adult coral-specific genes likely involved in coral-specific functions such as symbiosis and calcification.

Highly sensitive molecular diagnosis of prostate cancer using surplus material washed off from biopsy needles

PubMed Central

Bermudo, R; Abia, D; Mozos, A; García-Cruz, E; Alcaraz, A; Ortiz, Á R; Thomson, T M; Fernández, P L

2011-01-01

Introduction: Currently, final diagnosis of prostate cancer (PCa) is based on histopathological analysis of needle biopsies, but this process often bears uncertainties due to small sample size, tumour focality and pathologist's subjective assessment. Methods: Prostate cancer diagnostic signatures were generated by applying linear discriminant analysis to microarray and real-time RT–PCR (qRT–PCR) data from normal and tumoural prostate tissue samples. Additionally, after removal of biopsy tissues, material washed off from transrectal biopsy needles was used for molecular profiling and discriminant analysis. Results: Linear discriminant analysis applied to microarray data for a set of 318 genes differentially expressed between non-tumoural and tumoural prostate samples produced 26 gene signatures, which classified the 84 samples used with 100% accuracy. To identify signatures potentially useful for the diagnosis of prostate biopsies, surplus material washed off from routine biopsy needles from 53 patients was used to generate qRT–PCR data for a subset of 11 genes. This analysis identified a six-gene signature that correctly assigned the biopsies as benign or tumoural in 92.6% of the cases, with 88.8% sensitivity and 96.1% specificity. Conclusion: Surplus material from prostate needle biopsies can be used for minimal-size gene signature analysis for sensitive and accurate discrimination between non-tumoural and tumoural prostates, without interference with current diagnostic procedures. This approach could be a useful adjunct to current procedures in PCa diagnosis. PMID:22009027

Epithelial Membrane Protein-2 is a Novel Therapeutic Target in Ovarian Cancer

PubMed Central

Fu, Maoyong; Maresh, Erin L.; Soslow, Robert A.; Alavi, Mohammad; Mah, Vei; Zhou, Qin; Iasonos, Alexia; Goodglick, Lee; Gordon, Lynn K.; Braun, Jonathan; Wadehra, Madhuri

2010-01-01

Purpose The tetraspan protein epithelial membrane protein-2 (EMP2) has been shown to regulate the surface display and signaling from select integrin pairs, and it was recently identified as a prognostic biomarker in human endometrial cancer. In this study, we assessed the role of EMP2 in human ovarian cancer. Experimental Design We examined the expression of EMP2 within a population of women with ovarian cancer using tissue microarray assay technology. We evaluated the efficacy of EMP2-directed antibody therapy using a fully human recombinant bivalent antibody fragment (diabody) in vitro and ovarian cancer xenograft models in vivo. Results EMP2 was found to be highly expressed in over 70% of serous and endometrioid ovarian tumors compared to non-malignant ovarian epithelium using a human ovarian cancer tissue microarray. Using anti-EMP2 diabody, we evaluated the in vitro response of 9 human ovarian cancer cell lines with detectable EMP2 expression. Treatment of human ovarian cancer cell lines with anti-EMP2 diabodies induced cell death and retarded cell growth, and these response rates correlated with cellular EMP2 expression. We next assessed the effects of anti-EMP2 diabodies in mice bearing xenografts from the ovarian endometrioid carcinoma cell line OVCAR5. Anti-EMP2 diabodies significantly suppressed tumor growth and induced cell death in OVCAR5 xenografts. Conclusions These findings indicate that EMP2 is expressed in the majority of ovarian tumors and it may be a feasible target in vivo. PMID:20670949

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer.

PubMed

Wendel, Ulrika; Persson, Nina; Risinger, Christian; Bengtsson, Eva; Nodin, Björn; Danielsson, Lena; Welinder, Charlotte; Nordin Fredrikson, Gunilla; Jansson, Bo; Blixt, Ola

2018-01-01

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer

PubMed Central

Wendel, Ulrika; Persson, Nina; Risinger, Christian; Bengtsson, Eva; Nodin, Björn; Danielsson, Lena; Welinder, Charlotte; Nordin Fredrikson, Gunilla

2018-01-01

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE. PMID:29420566

The clinicopathologic association of c-MET overexpression in Iranian gastric carcinomas; an immunohistochemical study of tissue microarrays.

PubMed

Sotoudeh, Kambiz; Hashemi, Forough; Madjd, Zahra; Sadeghipour, Alireza; Molanaei, Saadat; Kalantary, Elham

2012-05-28

c-MET is an oncogene protein that plays important role in gastric carcinogenesis and has been introduced as a prognostic marker and potential therapeutic target. The aim of this study was to evaluate the frequency of c-MET overexpression and its relationship with clinicopathological variables in gastric cancer of Iranian population using tissue microarray. In a cross sectional study, representative paraffin blocks of 130 patients with gastric carcinoma treated by curative gastrectomy during a 2 years period of 2008-2009 in two university hospitals in Tehran-Iran were collected in tissue microarray and c-MET expression was studied by immunohistochemical staining. Finally 124 cases were evaluated, constituted of 99 male and 25 female with the average age of 61.5 years. In 71% (88/124) of tumors, c-MET high expression was found. c-MET high expression was more associated with intestinal than diffuse tumor type (P = 0.04), deeper tumor invasion, pT3 and pT4 versus pT1 and pT2 (P = 0.014), neural invasion (P = 0.002) and advanced TNM staging, stage 3 and 4 versus stage 1 and2 (P = 0.044). The c-MET high expression was not associated with age, sex, tumor location, differentiation grade and distant metastasis, but relative associations with lymph node metastasis (P = 0.065) and vascular invasion (P = 0.078) were observed. c-MET oncogene protein was frequently overexpressed in Iranian gastric carcinomas and it was related to clinicopathological characteristics such as tumor type, depth of invasion, neural invasion and TNM staging. It can also support the idea that c-MET is a potential marker for target therapy in Iranian gastric cancer. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9744598757151429.

The clinical and prognostic value of polo-like kinase 1 in lung squamous cell carcinoma patients: immunohistochemical analysis

PubMed Central

Li, Hefei; Sun, Zhenqing; Guo, Qiang; Shi, Hongyun; Jia, Youchao

2017-01-01

Polo-like kinase 1 (PLK1) has been suggested to serve as an oncogene in most human cancers. The aim of our study is to present more evidence about the clinical and prognostic value of PLK1 in lung squamous cell carcinoma patients. The status of PLK1 was observed in lung adenocarcinoma, lung squamous cell carcinoma, and normal lung tissues through analyzing microarray dataset (GEO accession numbers: GSE1213 and GSE 3627). PLK1 mRNA and protein expressions were detected in lung squamous cell carcinoma and normal lung tissues by using quantitative real-time PCR (qRT-PCR) and immunohistochemistry. In our results, the levels of PLK1 in lung squamous cell carcinoma tissues were higher than that in lung adenocarcinoma tissues. Compared with paired adjacent normal lung tissues, the PLK1 expression was increased in lung squamous cell carcinoma tissues. Furthermore, high expression of PLK1 protein was correlated with differentiated degree, clinical stage, tumor size, lymph node metastasis, and distant metastasis. The univariate and multivariate analyses showed PLK1 protein high expression was an unfavorable prognostic biomarker for lung squamous cell carcinoma patients. In conclusion, high expression of PLK1 is associated with the aggressive progression and poor prognosis in lung squamous cell carcinoma patients. PMID:28724602

Evaluation of the testicular toxicity of prenatal exposure to bisphenol A based on microarray analysis combined with MeSH annotation.

PubMed

Tainaka, Hitoshi; Takahashi, Hikari; Umezawa, Masakazu; Tanaka, Hiromitsu; Nishimune, Yoshitake; Oshio, Shigeru; Takeda, Ken

2012-01-01

Bisphenol A (BPA) is known to be an endocrine disruptor that affects the development of reproductive system. The aim of the present study was to investigate a group of testicular genes dysregulated by prenatal exposure to BPA. Pregnant ICR mice were treated with BPA by subcutaneous administration on days 7 and 14 of pregnancy. Tissue and blood samples were collected from 6-week-old male offspring. Testes were subjected to gene expression analysis using a testis-specific microarray (Testis2), consisting of 2,482 mouse cDNA clones annotated with Medical Subject Headings (MeSH) terms indicative of testicular components and functions. To interpret the microarray data, we used the MeSH terms significantly associated with the altered genes. As a result, MeSH terms related to androgens and Sertoli cells were extracted in BPA-treated groups. Among the genes related to Sertoli cells, downregulation of Msi1h, Ncoa1, Nid1, Hspb2, and Gata6 were detected in the testis of mice treated with BPA (twice administered 50 mg/kg). The MeSH terms associated with this group of genes may provide useful means to interpret the testicular toxicity of BPA. This article concludes that prenatal BPA exposure downregulates expression of genes associated with Sertoli cell function and affects the reproductive function of male offspring. Additionally, a method using MeSH to extract a group of genes was useful for predicting the testicular and reproductive toxicity of prenatal BPA exposure.

Effect of energy restriction and physical exercise intervention on phenotypic flexibility as examined by transcriptomics analyses of mRNA from adipose tissue and whole body magnetic resonance imaging.

PubMed

Lee, Sindre; Norheim, Frode; Langleite, Torgrim M; Noreng, Hans J; Storås, Trygve H; Afman, Lydia A; Frost, Gary; Bell, Jimmy D; Thomas, E Louise; Kolnes, Kristoffer J; Tangen, Daniel S; Stadheim, Hans K; Gilfillan, Gregor D; Gulseth, Hanne L; Birkeland, Kåre I; Jensen, Jørgen; Drevon, Christian A; Holen, Torgeir

2016-11-01

Overweight and obesity lead to changes in adipose tissue such as inflammation and reduced insulin sensitivity. The aim of this study was to assess how altered energy balance by reduced food intake or enhanced physical activity affect these processes. We studied sedentary subjects with overweight/obesity in two intervention studies, each lasting 12 weeks affecting energy balance either by energy restriction (~20% reduced intake of energy from food) in one group, or by enhanced energy expenditure due to physical exercise (combined endurance- and strength-training) in the other group. We monitored mRNA expression by microarray and mRNA sequencing from adipose tissue biopsies. We also measured several plasma parameters as well as fat distribution with magnetic resonance imaging and spectroscopy. Comparison of microarray and mRNA sequencing showed strong correlations, which were also confirmed using RT-PCR In the energy restricted subjects (body weight reduced by 5% during a 12 weeks intervention), there were clear signs of enhanced lipolysis as monitored by mRNA in adipose tissue as well as plasma concentration of free-fatty acids. This increase was strongly related to increased expression of markers for M1-like macrophages in adipose tissue. In the exercising subjects (glucose infusion rate increased by 29% during a 12-week intervention), there was a marked reduction in the expression of markers of M2-like macrophages and T cells, suggesting that physical exercise was especially important for reducing inflammation in adipose tissue with insignificant reduction in total body weight. Our data indicate that energy restriction and physical exercise affect energy-related pathways as well as inflammatory processes in different ways, probably related to macrophages in adipose tissue. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Recent progress in making protein microarray through BioLP

NASA Astrophysics Data System (ADS)

Yang, Rusong; Wei, Lian; Feng, Ying; Li, Xiujian; Zhou, Quan

2017-02-01

Biological laser printing (BioLP) is a promising biomaterial printing technique. It has the advantage of high resolution, high bioactivity, high printing frequency and small transported liquid amount. In this paper, a set of BioLP device is design and made, and protein microarrays are printed by this device. It's found that both laser intensity and fluid layer thickness have an influence on the microarrays acquired. Besides, two kinds of the fluid layer coating methods are compared, and the results show that blade coating method is better than well-coating method in BioLP. A microarray of 0.76pL protein microarray and a "NUDT" patterned microarray are printed to testify the printing ability of BioLP.

RNA sequencing: current and prospective uses in metabolic research.

PubMed

Vikman, Petter; Fadista, Joao; Oskolkov, Nikolay

2014-10-01

Previous global RNA analysis was restricted to known transcripts in species with a defined transcriptome. Next generation sequencing has transformed transcriptomics by making it possible to analyse expressed genes with an exon level resolution from any tissue in any species without any a priori knowledge of which genes that are being expressed, splice patterns or their nucleotide sequence. In addition, RNA sequencing is a more sensitive technique compared with microarrays with a larger dynamic range, and it also allows for investigation of imprinting and allele-specific expression. This can be done for a cost that is able to compete with that of a microarray, making RNA sequencing a technique available to most researchers. Therefore RNA sequencing has recently become the state of the art with regards to large-scale RNA investigations and has to a large extent replaced microarrays. The only drawback is the large data amounts produced, which together with the complexity of the data can make a researcher spend far more time on analysis than performing the actual experiment. © 2014 Society for Endocrinology.

Microarray-based characterization of differential gene expression during vocal fold wound healing in rats

PubMed Central

Welham, Nathan V.; Ling, Changying; Dawson, John A.; Kendziorski, Christina; Thibeault, Susan L.; Yamashita, Masaru

2015-01-01

The vocal fold (VF) mucosa confers elegant biomechanical function for voice production but is susceptible to scar formation following injury. Current understanding of VF wound healing is hindered by a paucity of data and is therefore often generalized from research conducted in skin and other mucosal systems. Here, using a previously validated rat injury model, expression microarray technology and an empirical Bayes analysis approach, we generated a VF-specific transcriptome dataset to better capture the system-level complexity of wound healing in this specialized tissue. We measured differential gene expression at 3, 14 and 60 days post-injury compared to experimentally naïve controls, pursued functional enrichment analyses to refine and add greater biological definition to the previously proposed temporal phases of VF wound healing, and validated the expression and localization of a subset of previously unidentified repair- and regeneration-related genes at the protein level. Our microarray dataset is a resource for the wider research community and has the potential to stimulate new hypotheses and avenues of investigation, improve biological and mechanistic insight, and accelerate the identification of novel therapeutic targets. PMID:25592437

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

PubMed

Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

2007-01-24

An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli.

PubMed

Geue, Lutz; Stieber, Bettina; Monecke, Stefan; Engelmann, Ines; Gunzer, Florian; Slickers, Peter; Braun, Sascha D; Ehricht, Ralf

2014-08-01

In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A systems biology approach to the pathogenesis of obesity-related nonalcoholic fatty liver disease using reverse phase protein microarrays for multiplexed cell signaling analysis.

PubMed

Calvert, Valerie S; Collantes, Rochelle; Elariny, Hazem; Afendy, Arian; Baranova, Ancha; Mendoza, Michael; Goodman, Zachary; Liotta, Lance A; Petricoin, Emanuel F; Younossi, Zobair M

2007-07-01

Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Omental adipose tissue, a biologically active organ secreting adipokines and cytokines, may play a role in the development of NAFLD. We tested this hypothesis with reverse-phase protein microarrays (RPA) for multiplexed cell signaling analysis of adipose tissue from patients with NAFLD. Omental adipose tissue was obtained from 99 obese patients. Liver biopsies obtained at the time of surgery were all read by the same hepatopathologist. Adipose tissue was exposed to rapid pressure cycles to extract protein lysates. RPA was used to investigate intracellular signaling. Analysis of 54 different kinase substrates and cell signaling endpoints showed that an insulin signaling pathway is deranged in different locations in NAFLD patients. Furthermore, components of insulin receptor-mediated signaling differentiate most of the conditions on the NAFLD spectrum. For example, PKA (protein kinase A) and AKT/mTOR (protein kinase B/mammalian target of rapamycin) pathway derangement accurately discriminates patients with NASH from those with the non-progressive forms of NAFLD. PKC (protein kinase C) delta, AKT, and SHC phosphorylation changes occur in patients with simple steatosis. Amounts of the FKHR (forkhead factor Foxo1)phosphorylated at S256 residue were significantly correlated with AST/ALT ratio in all morbidly obese patients. Furthermore, amounts of cleaved caspase 9 and pp90RSK S380 were positively correlated in patients with NASH. Specific insulin pathway signaling events are altered in the adipose tissue of patients with NASH compared with patients with nonprogressive forms of NAFLD. These findings provide evidence for the role of omental fat in the pathogenesis, and potentially, the progression of NAFLD.

Thrombus organization and healing in an experimental aneurysm model. Part II. The effect of various types of bioactive bioabsorbable polymeric coils.

PubMed

Yuki, Ichiro; Lee, Daniel; Murayama, Yuichi; Chiang, Alexander; Vinters, Harry V; Nismmura, Ichiro; Wang, Chiachien J; Ishii, Akira; Wu, Benjamin M; Viñuela, Fernando

2007-07-01

Bioabsorbable polymeric material coils are being used in the endovascular treatment of aneurysms to achieve better thrombus organization than is possible using bare platinum coils. We used immunohistochemical and molecular biological analysis techniques in experimental aneurysms implanted with three different bioabsorbable polymer coils and platinum coils. The degradation kinetics of nine polymer candidates for further analysis were first analyzed in vitro, and three materials with different degradation rates were selected. Seventy-four aneurysms were created in 37 swine using the venous pouch technique. The aneurysms were surgically implanted with one of the materials as follows (time points = 3, 7, and 14 days): Group 1, Guglielmi detachable coils (platinum); Group 2, Polysorb (90:10 polyglycolic acid [PGA]/polylactic acid); Group 3, Maxon (PGA/trimethylene carbonate); and Group 4, poly-l-lactic acid. Histological, immunohistochemical, and cDNA microarray analyses were performed on tissue specimens. Groups 1 and 4 showed minimal inflammatory response adjacent to the coil mass. In Group 2, Polysorb elicited a unique, firm granulation tissue that accelerated intraaneurysmal thrombus organization. In Group 3 intermediate inflammatory reactions were seen. Microarray analysis with Expression Analysis Sytematic Explorer software showed functional-cluster-gene activation to be increased at Day 7, preceding the histologic manifestation of polymer-induced granulation tissue at Day 14. A profile of expression changes in cytokine-related and extracellular membrane-related genes was compiled. Degradation speed was not the only factor determining the strength of the biological response. Polysorb induced an early, unique granulation tissue that conferred greater mechanical strength to the intraaneurysmal coilthrombus complex. Enhancing the formation of this polymer-induced granulation tissue may provide a new direction for improving long-term anatomical outcomes in cases involving aneurysms embolized with detachable coils.

Polymer microarray technology for stem cell engineering

PubMed Central

Coyle, Robert; Jia, Jia; Mei, Ying

2015-01-01

Stem cells hold remarkable promise for applications in tissue engineering and disease modeling. During the past decade, significant progress has been made in developing soluble factors (e.g., small molecules and growth factors) to direct stem cells into a desired phenotype. However, the current lack of suitable synthetic materials to regulate stem cell activity has limited the realization of the enormous potential of stem cells. This can be attributed to a large number of materials properties (e.g., chemical structures and physical properties of materials) that can affect stem cell fate. This makes it challenging to design biomaterials to direct stem cell behavior. To address this, polymer microarray technology has been developed to rapidly identify materials for a variety of stem cell applications. In this article, we summarize recent developments in polymer array technology and their applications in stem cell engineering. Statement of significance Stem cells hold remarkable promise for applications in tissue engineering and disease modeling. In the last decade, significant progress has been made in developing chemically defined media to direct stem cells into a desired phenotype. However, the current lack of the suitable synthetic materials to regulate stem cell activities has been limiting the realization of the potential of stem cells. This can be attributed to the number of variables in material properties (e.g., chemical structures and physical properties) that can affect stem cells. Polymer microarray technology has shown to be a powerful tool to rapidly identify materials for a variety of stem cell applications. Here we summarize recent developments in polymer array technology and their applications in stem cell engineering. PMID:26497624

Placental S100 (S100P) and GATA3: markers for transitional epithelium and urothelial carcinoma discovered by complementary DNA microarray.

PubMed

Higgins, John P T; Kaygusuz, Gulsah; Wang, Lingli; Montgomery, Kelli; Mason, Veronica; Zhu, Shirley X; Marinelli, Robert J; Presti, Joseph C; van de Rijn, Matt; Brooks, James D

2007-05-01

The morphologic distinction between prostate and urothelial carcinoma can be difficult. To identify novel diagnostic markers that may aid in the differential diagnosis of prostate versus urothelial carcinoma, we analyzed expression patterns in prostate and bladder cancer tissues using complementary DNA microarrays. Together with our prior studies on renal neoplasms and normal kidney, these studies suggested that the gene for placental S100 (S100P) is specifically expressed in benign and malignant urothelial cells. Using tissue microarrays, a polyclonal antiserum against S100P protein stained 86% of 295 urothelial carcinomas while only 3% of 260 prostatic adenocarcinomas and 1% of 133 renal cell carcinomas stained. A commercially available monoclonal antibody against S100P stained 78% of 300 urothelial carcinomas while only 2% of 256 prostatic adenocarcinomas and none of 137 renal cell carcinomas stained. A second gene, GATA3, also showed high level expression in urothelial tumors by cDNA array. A commercially available monoclonal antibody against GATA3 stained 67% of 308 urothelial carcinomas, but none of the prostate or renal carcinomas. For comparison, staining was also performed for p63 and cytokeratin 5/6. p63 stained 87% of urothelial carcinomas whereas CK5/6 stained 54%. Importantly, when S100P and p63 were combined 95% of urothelial carcinomas were labeled by one or both markers. We conclude that the detection of S100P and GATA3 protein expression may help distinguish urothelial carcinomas from other genitourinary neoplasms that enter into the differential diagnosis.

Upregulated expression of La ribonucleoprotein domain family member 6 and collagen type I gene following water-filtered broad-spectrum near-infrared irradiation in a 3-dimensional human epidermal tissue culture model as revealed by microarray analysis.

PubMed

Tanaka, Yohei; Nakayama, Jun

2018-05-01

Water-filtered broad-spectrum near-infrared irradiation can induce various biological effects, as our previous clinical, histological, and biochemical investigations have shown. However, few studies that examined the changes thus induced in gene expression. The aim was to investigate the changes in gene expression in a 3-dimensional reconstructed epidermal tissue culture exposed to water-filtered broad-spectrum near-infrared irradiation. DNA microarray and quantitative real-time polymerase chain reaction (PCR) analysis was used to assess gene expression levels in a 3-dimensional reconstructed epidermal model composed of normal human epidermal cells exposed to water-filtered broad-spectrum near-infrared irradiation. The water filter allowed 1000-1800 nm wavelengths and excluded 1400-1500 nm wavelengths, and cells were exposed to 5 or 10 rounds of near-infrared irradiation at 10 J/cm 2 . A DNA microarray with over 50 000 different probes showed 18 genes that were upregulated or downregulated by at least twofold after irradiation. Quantitative real-time PCR revealed that, relative to control cells, the gene encoding La ribonucleoprotein domain family member 6 (LARP6), which regulates collagen expression, was significantly and dose-dependently upregulated (P < 0.05) by water-filtered broad-spectrum near-infrared exposure. Gene encoding transcripts of collagen type I were significantly upregulated compared with controls (P < 0.05). This study demonstrates the ability of water-filtered broad-spectrum near-infrared irradiation to stimulate the production of type I collagen. © 2017 The Australasian College of Dermatologists.

Enhancing Results of Microarray Hybridizations Through Microagitation

PubMed Central

Toegl, Andreas; Kirchner, Roland; Gauer, Christoph; Wixforth, Achim

2003-01-01

Protein and DNA microarrays have become a standard tool in proteomics/genomics research. In order to guarantee fast and reproducible hybridization results, the diffusion limit must be overcome. Surface acoustic wave (SAW) micro-agitation chips efficiently agitate the smallest sample volumes (down to 10 μL and below) without introducing any dead volume. The advantages are reduced reaction time, increased signal-to-noise ratio, improved homogeneity across the microarray, and better slide-to-slide reproducibility. The SAW micromixer chips are the heart of the Advalytix ArrayBooster, which is compatible with all microarrays based on the microscope slide format. PMID:13678150

Living Cell Microarrays: An Overview of Concepts

PubMed Central

Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

2016-01-01

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

PubMed Central

van Huet, Ramon A. C.; Pierrache, Laurence H.M.; Meester-Smoor, Magda A.; Klaver, Caroline C.W.; van den Born, L. Ingeborgh; Hoyng, Carel B.; de Wijs, Ilse J.; Collin, Rob W. J.; Hoefsloot, Lies H.

2015-01-01

Purpose To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon–intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis. PMID:25999674

Transcriptomic Analysis Using Olive Varieties and Breeding Progenies Identifies Candidate Genes Involved in Plant Architecture

PubMed Central

González-Plaza, Juan J.; Ortiz-Martín, Inmaculada; Muñoz-Mérida, Antonio; García-López, Carmen; Sánchez-Sevilla, José F.; Luque, Francisco; Trelles, Oswaldo; Bejarano, Eduardo R.; De La Rosa, Raúl; Valpuesta, Victoriano; Beuzón, Carmen R.

2016-01-01

Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Little is known about how plant architecture is genetically determined in olive, were most of the existing varieties are traditional with an architecture poorly suited for modern growing and harvesting systems. In the present study, we have carried out microarray analysis of meristematic tissue to compare expression profiles of olive varieties displaying differences in architecture, as well as seedlings from their cross pooled on the basis of their sharing architecture-related phenotypes. The microarray used, previously developed by our group has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. Varieties with distinct architecture phenotypes and individuals from segregating progenies displaying opposite architecture features were used to link phenotype to expression. Here, we identify 2252 differentially expressed genes (DEGs) associated to differences in plant architecture. Microarray results were validated by quantitative RT-PCR carried out on genes with functional annotation likely related to plant architecture. Twelve of these genes were further analyzed in individual seedlings of the corresponding pool. We also examined Arabidopsis mutants in putative orthologs of these targeted candidate genes, finding altered architecture for most of them. This supports a functional conservation between species and potential biological relevance of the candidate genes identified. This study is the first to identify genes associated to plant architecture in olive, and the results obtained could be of great help in future programs aimed at selecting phenotypes adapted to modern cultivation practices in this species. PMID:26973682

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit

PubMed Central

Janssen, Bart J; Thodey, Kate; Schaffer, Robert J; Alba, Rob; Balakrishnan, Lena; Bishop, Rebecca; Bowen, Judith H; Crowhurst, Ross N; Gleave, Andrew P; Ledger, Susan; McArtney, Steve; Pichler, Franz B; Snowden, Kimberley C; Ward, Shayna

2008-01-01

Background Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45–55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. Results Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. Conclusion Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development. PMID:18279528

West Nile Virus Infection in Killer Whale, Texas, USA, 2007

PubMed Central

Wu, Guang; Anderson, Mark; Dalton, Les; Nilson, Erika; Wang, David

2011-01-01

In 2007, nonsuppurative encephalitis was identified in a killer whale at a Texas, USA, marine park. Panviral DNA microarray of brain tissue suggested West Nile virus (WNV); WNV was confirmed by reverse transcription PCR and sequencing. Immunohistochemistry demonstrated WNV antigen within neurons. WNV should be considered in cases of encephalitis in cetaceans. PMID:21801643

The civRT operon is important for Campylobacter jejuni strain 81-176 host cell interactions through regulation of the formate dehydrogenase operon

USDA-ARS?s Scientific Manuscript database

C. jejuni colonizes the intestinal mucosa, and the severity of disease in different strains is correlated with host cell interaction and invasion. A microarray screen to identify genes differentially regulated during C. jejuni interaction with tissue culture cells revealed the up-regulation of a two...

Crowdsourcing for translational research: analysis of biomarker expression using cancer microarrays

PubMed Central

Lawson, Jonathan; Robinson-Vyas, Rupesh J; McQuillan, Janette P; Paterson, Andy; Christie, Sarah; Kidza-Griffiths, Matthew; McDuffus, Leigh-Anne; Moutasim, Karwan A; Shaw, Emily C; Kiltie, Anne E; Howat, William J; Hanby, Andrew M; Thomas, Gareth J; Smittenaar, Peter

2017-01-01

Background: Academic pathology suffers from an acute and growing lack of workforce resource. This especially impacts on translational elements of clinical trials, which can require detailed analysis of thousands of tissue samples. We tested whether crowdsourcing – enlisting help from the public – is a sufficiently accurate method to score such samples. Methods: We developed a novel online interface to train and test lay participants on cancer detection and immunohistochemistry scoring in tissue microarrays. Lay participants initially performed cancer detection on lung cancer images stained for CD8, and we measured how extending a basic tutorial by annotated example images and feedback-based training affected cancer detection accuracy. We then applied this tutorial to additional cancer types and immunohistochemistry markers – bladder/ki67, lung/EGFR, and oesophageal/CD8 – to establish accuracy compared with experts. Using this optimised tutorial, we then tested lay participants' accuracy on immunohistochemistry scoring of lung/EGFR and bladder/p53 samples. Results: We observed that for cancer detection, annotated example images and feedback-based training both improved accuracy compared with a basic tutorial only. Using this optimised tutorial, we demonstrate highly accurate (>0.90 area under curve) detection of cancer in samples stained with nuclear, cytoplasmic and membrane cell markers. We also observed high Spearman correlations between lay participants and experts for immunohistochemistry scoring (0.91 (0.78, 0.96) and 0.97 (0.91, 0.99) for lung/EGFR and bladder/p53 samples, respectively). Conclusions: These results establish crowdsourcing as a promising method to screen large data sets for biomarkers in cancer pathology research across a range of cancers and immunohistochemical stains. PMID:27959886

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera

PubMed Central

2014-01-01

Background WRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown. Results We identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress. Conclusions We identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape. PMID:24755338

Gene expression profile of patients with Mayer-Rokitansky-Küster-Hauser syndrome: new insights into the potential role of developmental pathways.

PubMed

Nodale, Cristina; Ceccarelli, Simona; Giuliano, Mariateresa; Cammarota, Marcella; D'Amici, Sirio; Vescarelli, Enrica; Maffucci, Diana; Bellati, Filippo; Panici, Pierluigi Benedetti; Romano, Ferdinando; Angeloni, Antonio; Marchese, Cinzia

2014-01-01

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS.

Gene Expression Profile of Patients with Mayer-Rokitansky-Küster-Hauser Syndrome: New Insights into the Potential Role of Developmental Pathways

PubMed Central

Giuliano, Mariateresa; Cammarota, Marcella; D’Amici, Sirio; Vescarelli, Enrica; Maffucci, Diana; Bellati, Filippo; Panici, Pierluigi Benedetti; Romano, Ferdinando; Angeloni, Antonio; Marchese, Cinzia

2014-01-01

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS. PMID:24608967

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

PubMed

Xi, Qian-Yun; Xiong, Yuan-Yan; Wang, Yuan-Mei; Cheng, Xiao; Qi, Qi-En; Shu, Gang; Wang, Song-Bo; Wang, Li-Na; Gao, Ping; Zhu, Xiao-Tong; Jiang, Qing-Yan; Zhang, Yong-Liang; Liu, Li

2015-01-01

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

Assessing differential expression in two-color microarrays: a resampling-based empirical Bayes approach.

PubMed

Li, Dongmei; Le Pape, Marc A; Parikh, Nisha I; Chen, Will X; Dye, Timothy D

2013-01-01

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth's ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth's parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth's parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.

Implementation of mutual information and bayes theorem for classification microarray data

NASA Astrophysics Data System (ADS)

Dwifebri Purbolaksono, Mahendra; Widiastuti, Kurnia C.; Syahrul Mubarok, Mohamad; Adiwijaya; Aminy Ma’ruf, Firda

2018-03-01

Microarray Technology is one of technology which able to read the structure of gen. The analysis is important for this technology. It is for deciding which attribute is more important than the others. Microarray technology is able to get cancer information to diagnose a person’s gen. Preparation of microarray data is a huge problem and takes a long time. That is because microarray data contains high number of insignificant and irrelevant attributes. So, it needs a method to reduce the dimension of microarray data without eliminating important information in every attribute. This research uses Mutual Information to reduce dimension. System is built with Machine Learning approach specifically Bayes Theorem. This theorem uses a statistical and probability approach. By combining both methods, it will be powerful for Microarray Data Classification. The experiment results show that system is good to classify Microarray data with highest F1-score using Bayesian Network by 91.06%, and Naïve Bayes by 88.85%.

ENO1 Overexpression in Pancreatic Cancer Patients and Its Clinical and Diagnostic Significance

PubMed Central

Yin, Hang; Wang, Lei

2018-01-01

We investigated in this study the expression of ENO1 in tissues and plasma of PDAC patients to evaluate its clinicopathological and diagnostic significance. ENO1 protein expression was detected in tissue microarray of human PDAC and adjacent noncancer tissues. Electrochemiluminescence immunoassay and amplified luminescent proximity homogeneous assay (AlphaLISA) were performed to measure CA19-9 and ENO1 concentration in plasma from PDAC patients and healthy controls. We demonstrated that ENO1 overexpression is positively correlated with clinical stage, lymph node metastasis, and poor prognosis of PDAC; ENO1 may function as a hopeful candidate diagnostic marker in combination with CA19-9 in PDAC diagnosis. PMID:29483925

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

EPA Science Inventory

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, ...

Subcutaneous and gonadal adipose tissue transcriptome differences in lean and obese female dogs.

PubMed

Grant, Ryan W; Vester Boler, Brittany M; Ridge, Tonya K; Graves, Thomas K; Swanson, Kelly S

2013-12-01

Canine obesity leads to shortened life span and increased disease incidence. Adipose tissue depots are known to have unique metabolic and gene expression profiles in rodents and humans, but few comparisons of depot gene expression have been performed in the dog. Using microarray technology, our objective was to identify differentially expressed genes and enriched functional pathways between subcutaneous and gonadal adipose of lean and obese dogs to better understand the pathogenesis of obesity in the dog. Because no depot × body weight status interactions were identified in the microarray data, depot differences were the primary focus. A total of 946 and 703 transcripts were differentially expressed (FDR P < 0.05) between gonadal and subcutaneous adipose tissue in obese and lean dogs respectively. Of the adipose depot-specific differences in gene expression, 162 were present in both lean and obese dogs, with the majority (85%) expressed in the same direction. Both lean and obese dog gene lists had enrichment of the complement and coagulation cascade and systemic lupus erythematosus pathways. Obese dogs had enrichment of lysosome, extracellular matrix-receptor interaction, renin-angiotensin system and hematopoietic cell lineage pathways. Lean dogs had enrichment of glutathione metabolism and synthesis and degradation of ketone bodies. We have identified a core set of genes differentially expressed between subcutaneous and gonadal adipose tissue in dogs regardless of body weight. These genes contribute to depot-specific differences in immune function, extracellular matrix remodeling and lysosomal function and may contribute to the physiological differences noted between depots. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Increased Expression of ALDH1A1 in Prostate Cancer is Correlated With Tumor Aggressiveness: A Tissue Microarray Study of Iranian Patients.

PubMed

Kalantari, Elham; Saadi, Faezeh H; Asgari, Mojgan; Shariftabrizi, Ahmad; Roudi, Raheleh; Madjd, Zahra

2017-09-01

Subpopulations of prostate cancer (PCa) cells expressing putative stem cell markers possess the ability to promote tumor growth, maintenance, and progression. This study aimed to evaluate the expression patterns and clinical significance of putative stem cell marker aldehyde dehydrogenase 1 A1 (ALDH1A1) in prostate tumor tissues. ALDH1A1 expression was examined in a well-defined series of prostate tissues, including 105 (68%) samples of PCa, 21 (13%) samples of high-grade prostatic intraepithelial neoplasia, and 31 (19%) samples of benign prostate hyperplasia, which were embedded in tissue microarray blocks. The correlation of ALDH1A1 expression with clinicopathologic parameters was also assessed. There was a significant difference between the expression level of ALDH1A1 in PCa compared with the high-grade prostatic intraepithelial neoplasia and benign prostate hyperplasia samples (P<0.001). PCa cells expressing ALDH1A1 were more often seen in samples with advanced Gleason score (P=0.05) and high serum prostate specific antigen level (P=0.02). In addition, a positive correlation was found between ALDH1A1 expression and primary tumor stage and regional lymph node involvement (P=0.04 and 0.03, respectively). The significant association between ALDH1A1 expressions with Gleason score indicates the potential role of this protein in PCa tumorigenesis and aggressive behavior; therefore, this cancer stem cell marker can be used as a promising candidate for targeted therapy of PCa, especially those with high Gleason score.

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

PubMed

McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

2015-07-01

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

Practical considerations of image analysis and quantification of signal transduction IHC staining.

PubMed

Grunkin, Michael; Raundahl, Jakob; Foged, Niels T

2011-01-01

The dramatic increase in computer processing power in combination with the availability of high-quality digital cameras during the last 10 years has fertilized the grounds for quantitative microscopy based on digital image analysis. With the present introduction of robust scanners for whole slide imaging in both research and routine, the benefits of automation and objectivity in the analysis of tissue sections will be even more obvious. For in situ studies of signal transduction, the combination of tissue microarrays, immunohistochemistry, digital imaging, and quantitative image analysis will be central operations. However, immunohistochemistry is a multistep procedure including a lot of technical pitfalls leading to intra- and interlaboratory variability of its outcome. The resulting variations in staining intensity and disruption of original morphology are an extra challenge for the image analysis software, which therefore preferably should be dedicated to the detection and quantification of histomorphometrical end points.

The tissue micro-array data exchange specification: a web based experience browsing imported data

PubMed Central

Nohle, David G; Hackman, Barbara A; Ayers, Leona W

2005-01-01

Background The AIDS and Cancer Specimen Resource (ACSR) is an HIV/AIDS tissue bank consortium sponsored by the National Cancer Institute (NCI) Division of Cancer Treatment and Diagnosis (DCTD). The ACSR offers to approved researchers HIV infected biologic samples and uninfected control tissues including tissue cores in micro-arrays (TMA) accompanied by de-identified clinical data. Researchers interested in the type and quality of TMA tissue cores and the associated clinical data need an efficient method for viewing available TMA materials. Because each of the tissue samples within a TMA has separate data including a core tissue digital image and clinical data, an organized, standard approach to producing, navigating and publishing such data is necessary. The Association for Pathology Informatics (API) extensible mark-up language (XML) TMA data exchange specification (TMA DES) proposed in April 2003 provides a common format for TMA data. Exporting TMA data into the proposed format offers an opportunity to implement the API TMA DES. Using our public BrowseTMA tool, we created a web site that organizes and cross references TMA lists, digital "virtual slide" images, TMA DES export data, linked legends and clinical details for researchers. Microsoft Excel® and Microsoft Word® are used to convert tabular clinical data and produce an XML file in the TMA DES format. The BrowseTMA tool contains Extensible Stylesheet Language Transformation (XSLT) scripts that convert XML data into Hyper-Text Mark-up Language (HTML) web pages with hyperlinks automatically added to allow rapid navigation. Results Block lists, virtual slide images, legends, clinical details and exports have been placed on the ACSR web site for 14 blocks with 1623 cores of 2.0, 1.0 and 0.6 mm sizes. Our virtual microscope can be used to view and annotate these TMA images. Researchers can readily navigate from TMA block lists to TMA legends and to clinical details for a selected tissue core. Exports for 11 blocks with 3812 cores from three other institutions were processed with the BrowseTMA tool. Fifty common data elements (CDE) from the TMA DES were used and 42 more created for site-specific data. Researchers can download TMA clinical data in the TMA DES format. Conclusion Virtual TMAs with clinical data can be viewed on the Internet by interested researchers using the BrowseTMA tool. We have organized our approach to producing, sorting, navigating and publishing TMA information to facilitate such review. We have converted Excel TMA data into TMA DES XML, and imported it and TMA DES XML from another institution into BrowseTMA to produce web pages that allow us to browse through the merged data. We proposed enhancements to the TMA DES as a result of this experience. We implemented improvements to the API TMA DES as a result of using exported data from several institutions. A document type definition was written for the API TMA DES (that optionally includes proposed enhancements). Independent validators can be used to check exports against the DTD (with or without the proposed enhancements). Linking tissue core images to readily navigable clinical data greatly improves the value of the TMA. PMID:16086837

A database for the analysis of immunity genes in Drosophila: PADMA database.

PubMed

Lee, Mark J; Mondal, Ariful; Small, Chiyedza; Paddibhatla, Indira; Kawaguchi, Akira; Govind, Shubha

2011-01-01

While microarray experiments generate voluminous data, discerning trends that support an existing or alternative paradigm is challenging. To synergize hypothesis building and testing, we designed the Pathogen Associated Drosophila MicroArray (PADMA) database for easy retrieval and comparison of microarray results from immunity-related experiments (www.padmadatabase.org). PADMA also allows biologists to upload their microarray-results and compare it with datasets housed within PADMA. We tested PADMA using a preliminary dataset from Ganaspis xanthopoda-infected fly larvae, and uncovered unexpected trends in gene expression, reshaping our hypothesis. Thus, the PADMA database will be a useful resource to fly researchers to evaluate, revise, and refine hypotheses.

A probabilistic framework for microarray data analysis: fundamental probability models and statistical inference.

PubMed

Ogunnaike, Babatunde A; Gelmi, Claudio A; Edwards, Jeremy S

2010-05-21

Gene expression studies generate large quantities of data with the defining characteristic that the number of genes (whose expression profiles are to be determined) exceed the number of available replicates by several orders of magnitude. Standard spot-by-spot analysis still seeks to extract useful information for each gene on the basis of the number of available replicates, and thus plays to the weakness of microarrays. On the other hand, because of the data volume, treating the entire data set as an ensemble, and developing theoretical distributions for these ensembles provides a framework that plays instead to the strength of microarrays. We present theoretical results that under reasonable assumptions, the distribution of microarray intensities follows the Gamma model, with the biological interpretations of the model parameters emerging naturally. We subsequently establish that for each microarray data set, the fractional intensities can be represented as a mixture of Beta densities, and develop a procedure for using these results to draw statistical inference regarding differential gene expression. We illustrate the results with experimental data from gene expression studies on Deinococcus radiodurans following DNA damage using cDNA microarrays. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Using microarrays to identify positional candidate genes for QTL: the case study of ACTH response in pigs.

PubMed

Jouffe, Vincent; Rowe, Suzanne; Liaubet, Laurence; Buitenhuis, Bart; Hornshøj, Henrik; SanCristobal, Magali; Mormède, Pierre; de Koning, D J

2009-07-16

Microarray studies can supplement QTL studies by suggesting potential candidate genes in the QTL regions, which by themselves are too large to provide a limited selection of candidate genes. Here we provide a case study where we explore ways to integrate QTL data and microarray data for the pig, which has only a partial genome sequence. We outline various procedures to localize differentially expressed genes on the pig genome and link this with information on published QTL. The starting point is a set of 237 differentially expressed cDNA clones in adrenal tissue from two pig breeds, before and after treatment with adrenocorticotropic hormone (ACTH). Different approaches to localize the differentially expressed (DE) genes to the pig genome showed different levels of success and a clear lack of concordance for some genes between the various approaches. For a focused analysis on 12 genes, overlapping QTL from the public domain were presented. Also, differentially expressed genes underlying QTL for ACTH response were described. Using the latest version of the draft sequence, the differentially expressed genes were mapped to the pig genome. This enabled co-location of DE genes and previously studied QTL regions, but the draft genome sequence is still incomplete and will contain many errors. A further step to explore links between DE genes and QTL at the pathway level was largely unsuccessful due to the lack of annotation of the pig genome. This could be improved by further comparative mapping analyses but this would be time consuming. This paper provides a case study for the integration of QTL data and microarray data for a species with limited genome sequence information and annotation. The results illustrate the challenges that must be addressed but also provide a roadmap for future work that is applicable to other non-model species.

Microarray Analysis Reveals Increased Expression of Matrix Metalloproteases and Cytokines of Interleukin-20 Subfamily in the Kidneys of Neonate Rats Underwent Unilateral Ureteral Obstruction: A Potential Role of IL-24 in the Regulation of Inflammation and Tissue Remodeling.

PubMed

Pap, Domonkos; Sziksz, Erna; Kiss, Zoltán; Rokonay, Réka; Veres-Székely, Apor; Lippai, Rita; Takács, István Márton; Kis, Éva; Fekete, Andrea; Reusz, György; Szabó, Attila J; Vannay, Adam

2017-01-01

Congenital obstructive nephropathy (CON) is the main cause of pediatric chronic kidney diseases leading to renal fibrosis. High morbidity and limited treatment opportunities of CON urge the better understanding of the underlying molecular mechanisms. To identify the differentially expressed genes, microarray analysis was performed on the kidney samples of neonatal rats underwent unilateral ureteral obstruction (UUO). Microarray results were then validated by real-time RT-PCR and bioinformatics analysis was carried out to identify the relevant genes, functional groups and pathways involved in the pathomechanism of CON. Renal expression of matrix metalloproteinase (MMP)-12 and interleukin (IL)-24 were evaluated by real-time RT-PCR, flow cytometry and immunohistochemical analysis. Effect of the main profibrotic factors on the expression of MMP-12 and IL-24 was investigated on HK-2 and HEK-293 cell lines. Finally, the effect of IL-24 treatment on the expression of pro-inflammatory cytokines and MMPs were tested in vitro. Microarray analysis revealed 880 transcripts showing >2.0-fold change following UUO, enriched mainly in immune response related processes. The most up-regulated genes were MMPs and members of IL-20 cytokine subfamily, including MMP-3, MMP-7, MMP-12, IL-19 and IL-24. We found that while TGF-β treatment inhibits the expression of MMP-12 and IL-24, H2O2 or PDGF-B treatment induce the epithelial expression of MMP-12. We demonstrated that IL-24 treatment decreases the expression of IL-6 and MMP-3 in the renal epithelial cells. This study provides an extensive view of UUO induced changes in the gene expression profile of the developing kidney and describes novel molecules, which may play significant role in the pathomechanism of CON. © 2017 The Author(s)Published by S. Karger AG, Basel.

Identification of the Key Genes and Pathways in Esophageal Carcinoma.

PubMed

Su, Peng; Wen, Shiwang; Zhang, Yuefeng; Li, Yong; Xu, Yanzhao; Zhu, Yonggang; Lv, Huilai; Zhang, Fan; Wang, Mingbo; Tian, Ziqiang

2016-01-01

Objective . Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it. Methods . 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC. Results . A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis. Conclusion . The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

PubMed Central

Mitsios, Nick; Saka, Mohamad; Krupinski, Jerzy; Pennucci, Roberta; Sanfeliu, Coral; Wang, Qiuyu; Rubio, Francisco; Gaffney, John; Kumar, Pat; Kumar, Shant; Sullivan, Matthew; Slevin, Mark

2007-01-01

Background Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. Results Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. Conclusion Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents. PMID:17997827

A genome-wide 20 K citrus microarray for gene expression analysis

PubMed Central

Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

2008-01-01

Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. PMID:18598343

Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S; Jaing, C

2012-03-27

The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interimmore » report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.« less

An Introduction to MAMA (Meta-Analysis of MicroArray data) System.

PubMed

Zhang, Zhe; Fenstermacher, David

2005-01-01

Analyzing microarray data across multiple experiments has been proven advantageous. To support this kind of analysis, we are developing a software system called MAMA (Meta-Analysis of MicroArray data). MAMA utilizes a client-server architecture with a relational database on the server-side for the storage of microarray datasets collected from various resources. The client-side is an application running on the end user's computer that allows the user to manipulate microarray data and analytical results locally. MAMA implementation will integrate several analytical methods, including meta-analysis within an open-source framework offering other developers the flexibility to plug in additional statistical algorithms.

Neurofibromin Deficiency-Associated Transcriptional Dysregulation Suggests a Novel Therapy for Tibial Pseudoarthrosis in NF1

PubMed Central

Paria, Nandina; Cho, Tae-Joon; Choi, In Ho; Kamiya, Nobuhiro; Kayembe, Kay; Mao, Rong; Margraf, Rebecca L.; Obermosser, Gerlinde; Oxendine, Ila; Sant, David W.; Song, Mi Hyun; Stevenson, David A.; Viskochil, David H.; Wise, Carol A.; Kim, Harry K.W.; Rios, Jonathan J

2014-01-01

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic bi-allelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinosital-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant over-expression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing. PMID:24932921

Gene Profiling in Experimental Models of Eye Growth: Clues to Myopia Pathogenesis

PubMed Central

Stone, Richard A.; Khurana, Tejvir S.

2010-01-01

To understand the complex regulatory pathways that underlie the development of refractive errors, expression profiling has evaluated gene expression in ocular tissues of well-characterized experimental models that alter postnatal eye growth and induce refractive errors. Derived from a variety of platforms (e.g. differential display, spotted microarrays or Affymetrix GeneChips), gene expression patterns are now being identified in species that include chicken, mouse and primate. Reconciling available results is hindered by varied experimental designs and analytical/statistical features. Continued application of these methods offers promise to provide the much-needed mechanistic framework to develop therapies to normalize refractive development in children. PMID:20363242

C5b-9 Staining Correlates With Clinical and Tumor Stage in Gastric Adenocarcinoma.

PubMed

Chen, Jian; Yang, Wei-Jun; Sun, Hai-Jian; Yang, Xia; Wu, Yu-Zhang

2016-08-01

The complement system is a critical part of the immune response, acting in defense against viral infections, clearance of immune complexes, and maintenance of tissue homeostasis. Upregulated expression of the terminal complement complex, C5b-9, has been observed on various tumor cells, such as stomach carcinoma cells, and on cells in the necrotic regions of these tumors as well; however, whether and how C5b-9 is related to gastric cancer progression and severity remains unknown. In this study, human gastric adenocarcinoma (HGAC) tissues (n=47 cases) and patient-matched adjacent nontumoral parenchyma (n=20 cases) were evaluated by tissue microarray and immunohistochemistry. The HGAC tissues showed upregulated C5b-9 expression. Multinomial logistic regression and likelihood ratio testing showed that overexpression of C5b-9 in HGAC tissue was significantly correlated with clinical stage (P=0.007) and tumor stage (P=0.005), but not with tumor distant organ metastasis, lymphoid nodal status, sex, or age. Patients with late-stage gastric adenocarcinoma had a higher amount of tumor cells showing positive staining for C5b-9 than patients with early-stage disease. These results may help in diagnosis and assessment of disease severity of human gastric carcinoma.

Spatial changes in acid secretion from isolated stomach tissue using a pH-histamine sensing microarray.

PubMed

Bitziou, Eleni; O'Hare, Danny; Patel, Bhavik Anil

2010-03-01

The acid secretion mechanism can be studied by measuring a series of metabolic markers and neurotransmitters from in vitro isolated tissue. A microelectrode array was used to monitor proton concentration and histamine levels from isolated guinea pig stomach tissue. The device was partially modified using iridium oxide to form a series of pH sensors, whereas unmodified gold microelectrodes were used to measure the level of histamine in the gut. Real-time measurements in the presence of the H2-receptor antagonist ranitidine produced significant decreases in the overall Delta pH response, as expected. Also, a significant variation in the Delta pH response in between pH sensors was observed in the presence of pharmacological treatment due to structural features of the tissue. No significant differences in Delta i(H) were detected in the presence of ranitidine as expected. More significantly, clear variations in Delta pH responses between animals in control conditions and those in the presence of ranitidine was observed highlighting possible variation in parietal cell density and/or variations in tissue activity. These results identify great possibilities in applying these multi-sensing devices as a long-term stable personalised diagnostic tool for pharmacological screening and disease status.

Transcriptomic and bioinformatics analysis of the early time-course of the response to prostaglandin F2 alpha in the bovine corpus luteum

USDA-ARS?s Scientific Manuscript database

RNA expression analysis was performed on the corpus luteum tissue at five time points after prostaglandin F2 alpha treatment of midcycle cows using an Affymetrix Bovine Gene v1 Array. The normalized linear microarray data was uploaded to the NCBI GEO repository (GSE94069). Subsequent statistical ana...

Identification of Prostate Cancer Prognostic Markers

DTIC Science & Technology

2016-10-01

Technologies). For this, the oxygen consumption rate (OCR) in the PC-3 control and ECI1-overexpressing clones was measured following their maintenance...carnitine Carnitine β-oxydation Etomoxir Page 25 of 31 Figure 10: Mitochondrial Respiration in ECI1-overexpressing PC-3 Clones. Oxygen Consumption rate... FISH ), prognostic markers, biomarkers, tissue microarrays, autophagy 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES

Prostate Cancer Biorepository Network

DTIC Science & Technology

2017-10-01

Department of the Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704...clinical data including pathology and outcome data are annotated with the biospecimens. Specialized processing consists of tissue microarray design ...Months 1- 6): Completed in 1st quarter Task 5. Report on performance metrics: Ongoing (accrual reports are provided on quarterly basis) Task 6

Myogenin, AP2β, NOS-1, and HMGA2 are surrogate markers of fusion status in rhabdomyosarcoma: a report from the soft tissue sarcoma committee of the children's oncology group.

PubMed

Rudzinski, Erin R; Anderson, James R; Lyden, Elizabeth R; Bridge, Julia A; Barr, Frederic G; Gastier-Foster, Julie M; Bachmeyer, Karen; Skapek, Stephen X; Hawkins, Douglas S; Teot, Lisa A; Parham, David M

2014-05-01

Pediatric rhabdomyosarcoma (RMS) is traditionally classified on the basis of the histologic appearance into alveolar (ARMS) and embryonal (ERMS) subtypes. The majority of ARMS contain a PAX3-FOXO1 or PAX7-FOXO1 gene fusion, but about 20% do not. Intergroup Rhabdomyosarcoma Study stage-matched and group-matched ARMS typically behaves more aggressively than ERMS, but recent studies have shown that it is, in fact, the fusion status that drives the outcome for RMS. Gene expression microarray data indicate that several genes discriminate between fusion-positive and fusion-negative RMS with high specificity. Using tissue microarrays containing a series of both ARMS and ERMS, we identified a panel of 4 immunohistochemical markers-myogenin, AP2β, NOS-1, and HMGA2-which can be used as surrogate markers of fusion status in RMS. These antibodies provide an alternative to molecular methods for identification of fusion-positive RMS, particularly in cases in which there is scant or poor-quality material. In addition, these antibodies may be useful in fusion-negative ARMS as an indicator that a variant gene fusion may be present.

RHEB expression in fibroadenomas of the breast.

PubMed

Eom, Minseob; Han, Airi; Yi, Sang Yeop; Shin, John Junghun; Cui, Ying; Park, Kwang Hwa

2008-04-01

Although fibroadenoma is one of the most common types of benign breast tumor, genes specific to the tumor have not been identified. Microarrays were used to identify differentially expressed genes between fibroadenoma and infiltrating ductal carcinoma. The comparative expression of one of the identified genes, RAS homolog enriched in the brain (RHEB), was further explored using reverse transcriptase-polymerase chain reaction (RT-PCR). Microarray analysis was performed on tissue samples from five patients with fibroadenoma. In the fibroadenoma samples, the genes HDAC1, ROS1, TNFRSF10A, WASP2, TYRP1, WEE1, and RHEB were expressed at levels more than twofold higher than in the normal tissues. RT-PCR for RHEB indicated increased expression of RHEB in fibroadenoma compared to breast cancer. When studied with real-time PCR, the average RHEB/beta-actin ratio in fibroadenoma samples was 1.99, 2.46-fold greater than the average RHEB/beta-actin ratio in breast carcinoma of 0.81 (P < 0.01). Immunohistochemistry and PCR followed by microdissection shows increased expression of RHEB in epithelial cells compared to the stromal cells of fibroadenoma. Therefore, RHEB could be used cytopathologically to distinguish fibroadenoma from malignant breast carcinomas as a secondary diagnostic tool.

Transcriptome profiling reveals miR-9-3p as a novel tumor suppressor in gastric cancer.

PubMed

Meng, Qingshun; Xiang, Longquan; Fu, Jingwei; Chu, Xianqun; Wang, Chunlin; Yan, Bingzheng

2017-06-06

It has been well established that microRNAs (miRNAs) play important roles in biological processes. To comprehensively measure the altered miRNA expression, we presented the miRNA expression profile of gastric cancer using microarray. We identified 33 miRNAs that were significantly differentially regulated in gastric specimens compared to adjacent normal tissues, among which miR-9-3p expression are significantly down-regulated in gastric cancers. Next, a cohort of 100 gastric cancer tissues and matched normal tissues were enrolled. Kaplan-Meier and multivariate Cox survival analyses were applied to evaluate the prognostic value of miR-9-3p expression, and the result showed that patients with lower miR-9-3p expression level have significantly poorer overall survival. The expression level of miR-9-3p has been proved to be an independent prognostic factor for 5-year overall survival. Furthermore, the result indicated that over-expression of miR-9-3p can inhibit gastric cancer cell invasion. Taken together, our results suggested that miR-9-3p plays important role in tumor invasion, and these findings implicated the potential effects of miR-9-3p on prognosis of gastric cancer.

Alterations of protein glycosylation in embryonic stem cells during adipogenesis

PubMed Central

Liu, Wei; Wang, Yangyang; Rao, Yang; Yu, Hanjie; Cui, Jihong; Xie, Xin; Sun, Mei; Yin, Lu; Li, Hongmin; Chen, Fulin

2018-01-01

The understanding of adipose tissue development is crucial for the treatment of obesity-related diseases. Adipogenesis has been extensively investigated at the gene and protein levels in recent years. However, the alterations in protein glycosylation during this process remains unknown, particularly that of parthenogenetic embryonic stem cells (pESCs), a type of ESCs with low immunogenicity and no ethical concerns regarding their use. Protein glycosylation markedly affects cell growth and development, cell-to-cell communication, tumour growth and metastasis. In the present study, the adipogenic potentials of J1 ESCs and pESCs were first compared and the results demonstrated that pESCs had lower adipogenic potential compared with J1 ESCs. Lectin microarray was then used to screen the alteration of protein glycosylation during adipogenesis. The results revealed that protein modification of GlcNAc and α-1-2-fucosylation increased, whereas α-1-6-fucosylation, α-2-6-sialylation and α-1-6-mannosylation decreased in J1 ESCs and pESCs during this process. In addition, α-1-3-mannosylation decreased only in pESCs. Lectin histochemistry and quantitative polymerase chain reaction of glycosyltransferase confirmed the results obtained by lectin microarray. Therefore, protein glycosylation of ESCs was significantly altered during adipogenesis, indicating that protein glycosylation analysis is not only helpful for studying the mechanism of adipogenesis, but may also be used as a marker to monitor adipogenic development. PMID:29115405

Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis.

PubMed

Luo, Jun; Dunn, Thomas; Ewing, Charles; Sauvageot, Jurga; Chen, Yidong; Trent, Jeffrey; Isaacs, William

2002-05-15

Despite the high prevalence of benign prostatic hyperplasia (BPH) in the aging male, little is known regarding the etiology of this disease. A better understanding of the molecular etiology of BPH would be facilitated by a comprehensive analysis of gene expression patterns that are characteristic of benign growth in the prostate gland. Since genes differentially expressed between BPH and normal prostate tissues are likely to reflect underlying pathogenic mechanisms involved in the development of BPH, we performed comparative gene expression analysis using cDNA microarray technology to identify candidate genes associated with BPH. Total RNA was extracted from a set of 9 BPH specimens from men with extensive hyperplasia and a set of 12 histologically normal prostate tissues excised from radical prostatectomy specimens. Each of these 21 RNA samples was labeled with Cy3 in a reverse transcription reaction and cohybridized with a Cy5 labeled common reference sample to a cDNA microarray containing 6,500 human genes. Normalized fluorescent intensity ratios from each hybridization experiment were extracted to represent the relative mRNA abundance for each gene in each sample. Weighted gene and random permutation analyses were performed to generate a subset of genes with statistically significant differences in expression between BPH and normal prostate tissues. Semi-quantitative PCR analysis was performed to validate differential expression. A subset of 76 genes involved in a wide range of cellular functions was identified to be differentially expressed between BPH and normal prostate tissues. Semi-quantitative PCR was performed on 10 genes and 8 were validated. Genes consistently upregulated in BPH when compared to normal prostate tissues included: a restricted set of growth factors and their binding proteins (e.g. IGF-1 and -2, TGF-beta3, BMP5, latent TGF-beta binding protein 1 and -2); hydrolases, proteases, and protease inhibitors (e.g. neuropathy target esterase, MMP2, alpha-2-macroglobulin); stress response enzymes (e.g. COX2, GSTM5); and extracellular matrix molecules (e.g. laminin alpha 4 and beta 1, chondroitin sulfate proteoglycan 2, lumican). Genes consistently expressing less mRNA in BPH than in normal prostate tissues were less commonly observed and included the transcription factor KLF4, thrombospondin 4, nitric oxide synthase 2A, transglutaminase 3, and gastrin releasing peptide. We identified a diverse set of genes that are potentially related to benign prostatic hyperplasia, including genes both previously implicated in BPH pathogenesis as well as others not previously linked to this disease. Further targeted validation and investigations of these genes at the DNA, mRNA, and protein levels are warranted to determine the clinical relevance and possible therapeutic utility of these genes. Copyright 2002 Wiley-Liss, Inc.

Molecular differential diagnosis of follicular thyroid carcinoma and adenoma based on gene expression profiling by using formalin-fixed paraffin-embedded tissues

PubMed Central

2013-01-01

Background Differential diagnosis between malignant follicular thyroid cancer (FTC) and benign follicular thyroid adenoma (FTA) is a great challenge for even an experienced pathologist and requires special effort. Molecular markers may potentially support a differential diagnosis between FTC and FTA in postoperative specimens. The purpose of this study was to derive molecular support for differential post-operative diagnosis, in the form of a simple multigene mRNA-based classifier that would differentiate between FTC and FTA tissue samples. Methods A molecular classifier was created based on a combined analysis of two microarray datasets (using 66 thyroid samples). The performance of the classifier was assessed using an independent dataset comprising 71 formalin-fixed paraffin-embedded (FFPE) samples (31 FTC and 40 FTA), which were analysed by quantitative real-time PCR (qPCR). In addition, three other microarray datasets (62 samples) were used to confirm the utility of the classifier. Results Five of 8 genes selected from training datasets (ELMO1, EMCN, ITIH5, KCNAB1, SLCO2A1) were amplified by qPCR in FFPE material from an independent sample set. Three other genes did not amplify in FFPE material, probably due to low abundance. All 5 analysed genes were downregulated in FTC compared to FTA. The sensitivity and specificity of the 5-gene classifier tested on the FFPE dataset were 71% and 72%, respectively. Conclusions The proposed approach could support histopathological examination: 5-gene classifier may aid in molecular discrimination between FTC and FTA in FFPE material. PMID:24099521

Fasting induces a biphasic adaptive metabolic response in murine small intestine

PubMed Central

Sokolović, Milka; Wehkamp, Diederik; Sokolović, Aleksandar; Vermeulen, Jacqueline; Gilhuijs-Pederson, Lisa A; van Haaften, Rachel IM; Nikolsky, Yuri; Evelo, Chris TA; van Kampen, Antoine HC; Hakvoort, Theodorus BM; Lamers, Wouter H

2007-01-01

Background The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. Results Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. Conclusion The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover. PMID:17925015

Immunohistochemical expression of the mammalian target of rapamycin pathway in penile squamous cell carcinomas: a tissue microarray study of 112 cases.

PubMed

Chaux, Alcides; Munari, Enrico; Cubilla, Antonio L; Hicks, Jessica; Lecksell, Kristen; Burnett, Arthur L; Netto, George J

2014-05-01

The aim of this study was to evaluate the immunohistochemical expression of mammalian target of rapamycin (mTOR) pathway-related biomarkers in penile carcinomas, and to assess associations with histological type, histological grade, and human papillomavirus (HPV) infection. We built four tissue microarrays from 112 invasive penile squamous cell carcinomas, and evaluated the immunohistochemical expression of PTEN, phospho-AKT, phospho-mTOR, and phospho-S6. We found decreased or loss of PTEN expression in 87% of cases. Warty and/or basaloid carcinomas had a higher proportion of PTEN loss (P = 0.02), whereas keratinizing tumours showed higher levels of phospho-S6 (P = 0.009); phospho-AKT and phospho-mTOR levels were not significantly different between warty/basaloid and keratinizing carcinomas (P = 0.75 and P = 0.77, respectively). PTEN was not associated with histological grade (P = 0.18). Expression levels of phospho-S6 were significantly higher in low-grade tumours (P = 0.001), whereas expression levels of phospho-AKT and phospho-mTOR were slightly higher in high-grade tumours (P = 0.01 and P = 0.35, respectively). We did not find any association between HPV infection and mTOR markers (P ≥ 0.2 in all cases). Our results provide evidence of dysregulation of the mTOR pathway in penile carcinomas independently of HPV infection. Future clinical studies should further evaluate the prognostic and predictive usefulness of these markers in patients with penile cancer. © 2013 John Wiley & Sons Ltd.

The Porphyromonas gingivalis/Host Interactome Shows Enrichment in GWASdb Genes Related to Alzheimer's Disease, Diabetes and Cardiovascular Diseases

PubMed Central

Carter, Chris J.; France, James; Crean, StJohn; Singhrao, Sim K.

2017-01-01

Periodontal disease is of established etiology in which polymicrobial synergistic ecology has become dysbiotic under the influence of Porphyromonas gingivalis. Following breakdown of the host's protective oral tissue barriers, P. gingivalis migrates to developing inflammatory pathologies that associate with Alzheimer's disease (AD). Periodontal disease is a risk factor for cardiovascular disorders (CVD), type II diabetes mellitus (T2DM), AD and other chronic diseases, whilst T2DM exacerbates periodontitis. This study analyzed the relationship between the P. gingivalis/host interactome and the genes identified in genome-wide association studies (GWAS) for the aforementioned conditions using data from GWASdb (P < 1E-03) and, in some cases, from the NCBI/EBI GWAS database (P < 1E-05). Gene expression data from periodontitis or P. gingivalis microarray was compared to microarray datasets from the AD hippocampus and/or from carotid artery plaques. The results demonstrated that the host genes of the P. gingivalis interactome were significantly enriched in genes deposited in GWASdb genes related to cognitive disorders, AD and dementia, and its co-morbid conditions T2DM, obesity, and CVD. The P. gingivalis/host interactome was also enriched in GWAS genes from the more stringent NCBI-EBI database for AD, atherosclerosis and T2DM. The misregulated genes in periodontitis tissue or P. gingivalis infected macrophages also matched those in the AD hippocampus or atherosclerotic plaques. Together, these data suggest important gene/environment interactions between P. gingivalis and susceptibility genes or gene expression changes in conditions where periodontal disease is a contributory factor. PMID:29311898

The Porphyromonas gingivalis/Host Interactome Shows Enrichment in GWASdb Genes Related to Alzheimer's Disease, Diabetes and Cardiovascular Diseases.

PubMed

Carter, Chris J; France, James; Crean, StJohn; Singhrao, Sim K

2017-01-01

Periodontal disease is of established etiology in which polymicrobial synergistic ecology has become dysbiotic under the influence of Porphyromonas gingivalis . Following breakdown of the host's protective oral tissue barriers, P. gingivalis migrates to developing inflammatory pathologies that associate with Alzheimer's disease (AD). Periodontal disease is a risk factor for cardiovascular disorders (CVD), type II diabetes mellitus (T2DM), AD and other chronic diseases, whilst T2DM exacerbates periodontitis. This study analyzed the relationship between the P. gingivalis /host interactome and the genes identified in genome-wide association studies (GWAS) for the aforementioned conditions using data from GWASdb ( P < 1E-03) and, in some cases, from the NCBI/EBI GWAS database ( P < 1E-05). Gene expression data from periodontitis or P. gingivalis microarray was compared to microarray datasets from the AD hippocampus and/or from carotid artery plaques. The results demonstrated that the host genes of the P. gingivalis interactome were significantly enriched in genes deposited in GWASdb genes related to cognitive disorders, AD and dementia, and its co-morbid conditions T2DM, obesity, and CVD. The P. gingivalis /host interactome was also enriched in GWAS genes from the more stringent NCBI-EBI database for AD, atherosclerosis and T2DM. The misregulated genes in periodontitis tissue or P. gingivalis infected macrophages also matched those in the AD hippocampus or atherosclerotic plaques. Together, these data suggest important gene/environment interactions between P. gingivalis and susceptibility genes or gene expression changes in conditions where periodontal disease is a contributory factor.

Protective effect of bicyclol against bile duct ligation-induced hepatic fibrosis in rats

PubMed Central

Zhen, Yong-Zhan; Li, Na-Ren; He, Hong-Wei; Zhao, Shuang-Shuang; Zhang, Guang-Ling; Hao, Xiao-Fang; Shao, Rong-Guang

2015-01-01

AIM: To evaluate the protective effect of bicyclol against bile duct ligation (BDL)-induced hepatic fibrosis in rats. METHODS: Sprague-Dawley male rats underwent BDL and sham-operated animals were used as healthy controls. The BDL rats were divided into two groups which received sterilized PBS or bicyclol (100 mg/kg per day) orally for two consecutive weeks. Serum, urine and bile were collected for biochemical determinations. Liver tissues were collected for histological analysis and a whole genome oligonucleotide microarray assay. Reverse transcription-polymerase chain reaction and Western blotting were used to verify the expression of liver fibrosis-related genes. RESULTS: Treatment with bicyclol significantly reduced liver fibrosis and bile duct proliferation after BDL. The levels of alanine aminotransferase (127.7 ± 72.3 vs 230.4 ± 69.6, P < 0.05) and aspartate aminotransferase (696.8 ± 232.6 vs 1032.6 ± 165.8, P < 0.05) were also decreased by treatment with bicyclol in comparison to PBS. The expression changes of 45 fibrogenic genes and several fibrogenesis-related pathways were reversed by bicyclol in the microarray assay. Bicyclol significantly reduced liver mRNA and/or protein expression levels of collagen 1a1, matrix metalloproteinase 2, tumor necrosis factor, tissue inhibitors of metalloproteinases 2, transforming growth factor-β1 and α-smooth muscle actin. CONCLUSION: Bicyclol significantly attenuates BDL-induced liver fibrosis by reversing fibrogenic gene expression. These findings suggest that bicyclol might be an effective anti-fibrotic drug for the treatment of cholestatic liver disease. PMID:26109801

Influence of negative energy balance on cyclicity and fertility in the high producing dairy cow.

PubMed

Wathes, D C; Fenwick, M; Cheng, Z; Bourne, N; Llewellyn, S; Morris, D G; Kenny, D; Murphy, J; Fitzpatrick, R

2007-09-01

The peripartum period is of critical importance to subsequent health and fertility. Most cows enter a state of negative energy balance (NEB) associated with many metabolic changes which have carry over effects on the resumption and normality of estrous cyclicity and the success of subsequent inseminations. A dataset on 500 lactations explored the relationships between metabolic traits measured before and after calving with fertility. Stepwise multiple regression analysis showed that longer calving to conception intervals were associated with altered profiles of IGF-I, urea and body condition score. These relationships between metabolic profiles and fertility differed between first lactation cows (which are still growing but produce less milk) and mature animals. Early postpartum the liver undergoes extensive biochemical and morphological modifications to adapt to NEB, the uterus is extensively remodeled and must clear bacterial infections, and the ovary must resume ovulatory cycles. RNA isolated from liver and uterine tissues harvested 2 weeks postpartum from cows in mild (MNEB) and severe (SNEB) energy balance was used to screen the Affymetrix 23K bovine microarray. In liver, SNEB resulted in differential expression of key genes involved in lipid catabolism, gluconeogenesis, and the synthesis and stability of IGF-I. This was accompanied by reduced systemic concentrations of IGF-I which is likely to impact on ovarian function and early embryo development. Within endometrium, cows in SNEB showed histological evidence for higher levels of inflammation and the microarray analysis identified groups of differentially expressed genes involved in tissue remodeling and immune response. This may delay uterine repair after calving, likely contributing to the observed reduction in fertility.

Identification and functional analysis of microRNA in myometrium tissue from spontaneous preterm labor

PubMed Central

Tang, Yao; Ji, Hongjing; Liu, Haiyan; Gu, Weirong; Li, Xiaotian; Peng, Ting

2015-01-01

Spontaneous preterm labor is an important complication in perinatology characterized by early onset myometrium contractions leading to labor at preterm. However, the exact mechanism that maintain uterine quiescence and promote increased uterine contractility during labor were incompletely defined. MicroRNAs is a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level by binding the 3’ untranslated region of target mRNAs and play an important role in biological process and cellular functions. We hypothesized we could find differentially expressed microRNAs in the myometrium of women in spontaneous preterm labor. Thus, a microarray analysis of miRNAs of preterm myometrium was performed. 18 out of the 2006 detected microRNAs were found to be significantly dysregulated in myometrium in labor verse not in labor at preterm. Biological validation by quantitative real-time polymerase chain reaction confirms us a consistence rate of 83.3% (5 out of 6) with microarray analysis. The target genes for validated microRNAs were predicted by three algorithms (PicTar, TargetScan, and miRanda). Most of the potential targets of the miRNAs were relevant to positive regulation of cardiac muscle hypertrophy, reduction of cytosolic calcium ion concentration and relaxation of cardiac muscle as well as prostate cancer, adherents junction, regulation of actin cytoskeleton and regulation and other factor-regulated calcium reabsorption. Our result illustrates a characteristic microRNA profile in myometrium tissues and provides a new understanding of the process involved in spontaneous preterm labor. PMID:26722471

Comparison of estrogen receptor results from pathology reports with results from central laboratory testing.

PubMed

Collins, Laura C; Marotti, Jonathan D; Baer, Heather J; Tamimi, Rulla M

2008-02-06

We compared estrogen receptor (ER) assay results abstracted from pathology reports with ER results determined on the same specimens by a central laboratory with an immunohistochemical assay. Paraffin sections were cut from tissue microarrays containing 3093 breast cancer specimens from women enrolled in the Nurses' Health Study, 1851 of which had both pathology reports and tissue available for central laboratory testing. All sections were immunostained for ER at the same time. The original assays were biochemical for 1512 (81.7%) of the 1851 specimens, immunohistochemical for 336 (18.2%), and immunofluorescent for three (0.2%). ER results from pathology reports and repeat central laboratory testing were in agreement for 87.3% of specimens (1615 of the 1851 specimens; kappa statistic = 0.64, P < .001). When the comparison was restricted to the specimens for which the ER assays were originally performed by immunohistochemistry, the agreement rate increased to 92.3% of specimens (310 of the 336 specimens; kappa statistic = 0.78, P < .001). Thus, ER assay results from pathology reports appear to be a reasonable alternative to central laboratory ER testing for large, population-based studies of patients with breast cancer.

Gene Expression Profiling of the Hypoxia Signaling Pathway in Hypoxia-Inducible Factor 1α Null Mouse Embryonic Fibroblasts

PubMed Central

Vengellur, Ajith; Woods, Barbara G.; Ryan, Heather E.; Johnson, Randall S.; Lapres, John J.

2003-01-01

Hypoxia is defined as a deficiency of oxygen reaching the tissues of the body, and it plays a critical role in development and pathological conditions, such as cancer. Once tumors outgrow their blood supply, their central portion becomes hypoxic and the tumor stimulates angiogenesis through the activation of the hypoxia-inducible factors (HIFs). HIFs are transcription factors that are regulated in an oxygen-dependent manner by a group of prolyl hydroxylases (known as PHDs or HPHs). Our understanding of hypoxia signaling is limited by our incomplete knowledge of HIF target genes. cDNA microarrays and a cell line lacking a principal HIF protein, HIF1α, were used to identify a more complete set of hypoxia-regulated genes. The microarrays identified a group of 286 clones that were significantly influenced by hypoxia and 54 of these were coordinately regulated by cobalt chloride. The expression profile of HIF1α −/− cells also identified a group of downregulated genes encoding enzymes involved in protecting cells from oxidative stress, offering an explanation for the increased sensitivity of HIF1α −/− cells to agents that promote this type of response. The microarray studies confirmed the hypoxia-induced expression of the HIF regulating prolyl hydroxylase, PHD2. An analysis of the members of the PHD family revealed that they are differentially regulated by cobalt chloride and hypoxia. These results suggest that HIF1α is the predominant isoform in fibroblasts and that it regulates a wide battery of genes critical for normal cellular function and survival under various stresses. PMID:14686790

Long non-coding RNA ENST00462717 suppresses the proliferation, survival, and migration by inhibiting MDM2/MAPK pathway in glioma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Aiqin; Meng, Mingzhu; Zhao, Xiuhe

Gliomas are the most common and aggressive primary malignant tumor in the central nervous system, and requires new biomarkers and therapeutic methods. Long noncoding RNAs (lncRNAs) are important factors in numerous human diseases, including cancer. But studies on lncRNAs and gliomas are limited. In this study, we investigated the expression patterns of lncRNAs in 3 pairs of glioma samples and adjacent non-tumor tissues via microarray and selected the most down-regulated lnc00462717 to further verify its roles in glioma. We observed that decreased lnc00462717 expression was associated with the malignant status in glioma. In vitro experiment demonstrated that lnc00462717 overexpression suppressed gliomamore » cell proliferation, survival and migration while knockdown of lnc00462717 had an opposite result. Moreover, we identified MDM2 as a direct target of lnc00462717 and lnc00462717 played a role by partially regulating the MDM2/MAPK pathway. In conclusion, lnc00462717 may function in suppressing glioma cell proliferation, survival, migration and may potentially serve as a novel biomarker and therapeutic target for glioma. - Highlights: • Using microarray to investigate the expression patterns of lncRNAs in glioma. • Selecting the most down-regulated lnc00462717 via microarray to verify its roles. • Identifying MDM2 as a direct target of lnc00462717. • The mechanism of lnc00462717 regulating the MDM2/MAPK pathway. • lnc00462717 serve as a novel biomarker and therapeutic target for treating glioma.« less

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

PubMed

Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

2008-10-01

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

Form-Deprivation Myopia in Chick Induces Limited Changes in Retinal Gene Expression

PubMed Central

McGlinn, Alice M.; Baldwin, Donald A.; Tobias, John W.; Budak, Murat T.; Khurana, Tejvir S.; Stone, Richard A.

2007-01-01

Purpose Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick. Methods One-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription–polymerase chain reaction (qPCR) in independent biological replicates. Results Small changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II–related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated. Conclusions Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development. PMID:17652709

Microarray analysis identifies candidate genes for key roles in coral development

PubMed Central

Grasso, Lauretta C; Maindonald, John; Rudd, Stephen; Hayward, David C; Saint, Robert; Miller, David J; Ball, Eldon E

2008-01-01

Background Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired. Results Of 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization. Conclusion This study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemone Nematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease. PMID:19014561

Expansion of Multipotent Stem Cells from the Adult Human Brain

PubMed Central

Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

2013-01-01

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome

PubMed Central

Boqun, Xu; Xiaonan, Dai; YuGui, Cui; Lingling, Gao; Xue, Dai; Gao, Chao; Feiyang, Diao; Jiayin, Liu; Gao, Li; Li, Mei; Zhang, Yuan; Ma, Xiang

2013-01-01

Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS. PMID:23861679

Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome.

PubMed

Boqun, Xu; Xiaonan, Dai; Yugui, Cui; Lingling, Gao; Xue, Dai; Gao, Chao; Feiyang, Diao; Jiayin, Liu; Gao, Li; Li, Mei; Zhang, Yuan; Ma, Xiang

2013-01-01

Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS.

Comparing bacterial community composition between healthy and white plague-like disease states in Orbicella annularis using PhyloChip™ G3 microarrays

USGS Publications Warehouse

Kellogg, Christina A.; Piceno, Yvette M.; Tom, Lauren M.; DeSantis, Todd Z.; Gray, Michael A.; Zawada, David G.; Andersen, Gary L.

2013-01-01

Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes ‘white plague.’ PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral (Orbicella annularis, formerly of the genus Montastraea [1]) were collected from two geographically distinct areas, Dry Tortugas National Park and Virgin Islands National Park, to determine if there were biogeographic differences between the diseases. In fact, all diseased samples clustered together, however there was no consistent link to Aurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups (Actinobacteria, Firmicutes) comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™ data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state.

Comparing Bacterial Community Composition between Healthy and White Plague-Like Disease States in Orbicella annularis Using PhyloChip™ G3 Microarrays

PubMed Central

Kellogg, Christina A.; Piceno, Yvette M.; Tom, Lauren M.; DeSantis, Todd Z.; Gray, Michael A.; Zawada, David G.; Andersen, Gary L.

2013-01-01

Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes ‘white plague.’ PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral (Orbicella annularis, formerly of the genus Montastraea [1]) were collected from two geographically distinct areas, Dry Tortugas National Park and Virgin Islands National Park, to determine if there were biogeographic differences between the diseases. In fact, all diseased samples clustered together, however there was no consistent link to Aurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups (Actinobacteria, Firmicutes) comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state. PMID:24278181

Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model

PubMed Central

Tanaka, Yohei; Nakayama, Jun

2016-01-01

Background and objective Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. Materials and methods DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths. Results A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05). Conclusion We found that NIR irradiation induced the upregulated expression of EGFR in human corneal cells. Since over half of the solar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in turn eye damage. PMID:27536083

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima

PubMed Central

2011-01-01

Background Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. Results A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes. Conclusions This investigation has mapped the spatial distribution for over 2000 ESTs present on PmaxArray 1.0 with reference to specific locations of the mantle. Expression profile clusters have indicated at least five unique functioning zones in the mantle. Three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. A number of novel and known transcripts have been identified from these clusters. The development of PmaxArray 1.0, and the spatial map of its ESTs expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell. PMID:21936921

Characterization and simulation of cDNA microarray spots using a novel mathematical model

PubMed Central

Kim, Hye Young; Lee, Seo Eun; Kim, Min Jung; Han, Jin Il; Kim, Bo Kyung; Lee, Yong Sung; Lee, Young Seek; Kim, Jin Hyuk

2007-01-01

Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of spot images. Results We developed a governing equation of cDNA deposition during evaporation of a drop in the microarray spotting process. The governing equation included four parameters: the surface site density on the support, the extrapolated equilibrium constant for the binding of cDNA molecules with surface sites on glass slides, the macromolecular interaction factor, and the volume constant of a drop of cDNA solution. We simulated cDNA deposition from the single model equation by varying the value of the parameters. The morphology of the resulting cDNA deposit can be classified into three types: a doughnut shape, a peak shape, and a volcano shape. The spot morphology can be changed into a flat shape by varying the experimental conditions while considering the parameters of the governing equation of cDNA deposition. The four parameters were estimated by fitting the governing equation to the real microarray images. With the results of the simulation and the parameter estimation, the phenomenon of the formation of cDNA deposits in each type was investigated. Conclusion This study explains how various spot shapes can exist and suggests which parameters are to be adjusted for obtaining a good spot. This system is able to explore the cDNA microarray spotting process in a predictable, manageable and descriptive manner. We hope it can provide a way to predict the incidents that can occur during a real cDNA microarray experiment, and produce useful data for several research applications involving cDNA microarrays. PMID:18096047

Microarray RNA expression analysis of cerebral white matter lesions reveals changes in multiple functional pathways.

PubMed

Simpson, Julie E; Hosny, Ola; Wharton, Stephen B; Heath, Paul R; Holden, Hazel; Fernando, Malee S; Matthews, Fiona; Forster, Gill; O'Brien, John T; Barber, Robert; Kalaria, Raj N; Brayne, Carol; Shaw, Pamela J; Lewis, Claire E; Ince, Paul G

2009-02-01

White matter lesions (WML) in brain aging are linked to dementia and depression. Ischemia contributes to their pathogenesis but other mechanisms may contribute. We used RNA microarray analysis with functional pathway grouping as an unbiased approach to investigate evidence for additional pathogenetic mechanisms. WML were identified by MRI and pathology in brains donated to the Medical Research Council Cognitive Function and Ageing Study Cognitive Function and Aging Study. RNA was extracted to compare WML with nonlesional white matter samples from cases with lesions (WM[L]), and from cases with no lesions (WM[C]) using RNA microarray and pathway analysis. Functional pathways were validated for selected genes by quantitative real-time polymerase chain reaction and immunocytochemistry. We identified 8 major pathways in which multiple genes showed altered RNA transcription (immune regulation, cell cycle, apoptosis, proteolysis, ion transport, cell structure, electron transport, metabolism) among 502 genes that were differentially expressed in WML compared to WM[C]. In WM[L], 409 genes were altered involving the same pathways. Genes selected to validate this microarray data all showed the expected changes in RNA levels and immunohistochemical expression of protein. WML represent areas with a complex molecular phenotype. From this and previous evidence, WML may arise through tissue ischemia but may also reflect the contribution of additional factors like blood-brain barrier dysfunction. Differential expression of genes in WM[L] compared to WM[C] indicate a "field effect" in the seemingly normal surrounding white matter.

Microarray platform affords improved product analysis in mammalian cell growth studies

PubMed Central

Li, Lingyun; Migliore, Nicole; Schaefer, Eugene; Sharfstein, Susan T.; Dordick, Jonathan S.; Linhardt, Robert J.

2014-01-01

High throughput (HT) platforms serve as cost-efficient and rapid screening method for evaluating the effect of cell culture conditions and screening of chemicals. The aim of the current study was to develop a high-throughput cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/MSX CHO cell line, which produces a therapeutic monoclonal antibody, was examined using microarray system in conjunction with conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60 nl spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base media results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the high-throughput microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as, cell growth, metabolism and productivity. PMID:24227746

Far infrared promotes wound healing through activation of Notch1 signaling.

PubMed

Hsu, Yung-Ho; Lin, Yuan-Feng; Chen, Cheng-Hsien; Chiu, Yu-Jhe; Chiu, Hui-Wen

2017-11-01

The Notch signaling pathway is critically involved in cell proliferation, differentiation, development, and homeostasis. Far infrared (FIR) has an effect that promotes wound healing. However, the underlying molecular mechanisms are unclear. In the present study, we employed in vivo and HaCaT (a human skin keratinocyte cell line) models to elucidate the role of Notch1 signaling in FIR-promoted wound healing. We found that FIR enhanced keratinocyte migration and proliferation. FIR induced the Notch1 signaling pathway in HaCaT cells and in a microarray dataset from the Gene Expression Omnibus database. We next determined the mRNA levels of NOTCH1 in paired normal and wound skin tissues derived from clinical patients using the microarray dataset and Ingenuity Pathway Analysis software. The result indicated that the Notch1/Twist1 axis plays important roles in wound healing and tissue repair. In addition, inhibiting Notch1 signaling decreased the FIR-enhanced proliferation and migration. In a full-thickness wound model in rats, the wounds healed more rapidly and the scar size was smaller in the FIR group than in the light group. Moreover, FIR could increase Notch1 and Delta1 in skin tissues. The activation of Notch1 signaling may be considered as a possible mechanism for the promoting effect of FIR on wound healing. FIR stimulates keratinocyte migration and proliferation. Notch1 in keratinocytes has an essential role in FIR-induced migration and proliferation. NOTCH1 promotes TWIST1-mediated gene expression to assist wound healing. FIR might promote skin wound healing in a rat model. FIR stimulates keratinocyte migration and proliferation. Notch1 in keratinocytes has an essential role in FIR-induced migration and proliferation. NOTCH1 promotes TWIST1-mediated gene expression to assist wound healing. FIR might promote skin wound healing in a rat model.

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

NASA Technical Reports Server (NTRS)

Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr;

Effects of Lactobacillus fermented soymilk and soy yogurt on hepatic lipid accumulation in rats fed a cholesterol-free diet.

PubMed

Kitawaki, Ryoko; Nishimura, Yuko; Takagi, Naohiro; Iwasaki, Mitsuhiro; Tsuzuki, Kimiko; Fukuda, Mitsuru

2009-07-01

We examined the effects of lactic acid fermented soymilk, in which part of the soymilk was replaced with okara (soy yogurt), on plasma and hepatic lipid profiles in rats fed a cholesterol-free diet. Additionally, we investigated the effects of soy yogurt on hepatic gene expression in rats using DNA microarray analysis. Male Sprague-Dawley rats aged 5 weeks (n=5/group) were fed a control diet (AIN-93) or a test diet in which 20% of the diet was replaced by soy yogurt for 7 weeks. Soy yogurt consumption did not affect body weight or adipose tissue weight as compared with control diet. In the soy yogurt group, the liver weight and hepatic triglyceride content were significantly lower than the control group, and the level of plasma cholesterol was also lower. Furthermore, DNA microarray analysis indicated that soy yogurt ingestion down-regulated the expression of the SREBP-1 gene and enzymes related to lipogenesis in the rat liver, while expression of beta-oxidation-related genes was up-regulated. These results suggest that soy yogurt is beneficial in preventing hepatic lipid accumulation in rats.

Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

PubMed

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

2015-10-06

Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

Comparing transformation methods for DNA microarray data.

PubMed

Thygesen, Helene H; Zwinderman, Aeilko H

2004-06-17

When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include subtraction of an estimated background signal, subtracting the reference signal, smoothing (to account for nonlinear measurement effects), and more. Different authors use different approaches, and it is generally not clear to users which method they should prefer. We used the ratio between biological variance and measurement variance (which is an F-like statistic) as a quality measure for transformation methods, and we demonstrate a method for maximizing that variance ratio on real data. We explore a number of transformations issues, including Box-Cox transformation, baseline shift, partial subtraction of the log-reference signal and smoothing. It appears that the optimal choice of parameters for the transformation methods depends on the data. Further, the behavior of the variance ratio, under the null hypothesis of zero biological variance, appears to depend on the choice of parameters. The use of replicates in microarray experiments is important. Adjustment for the null-hypothesis behavior of the variance ratio is critical to the selection of transformation method.

Segment and Fit Thresholding: A New Method for Image Analysis Applied to Microarray and Immunofluorescence Data

PubMed Central

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M.; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E.; Allen, Peter J.; Sempere, Lorenzo F.; Haab, Brian B.

2016-01-01

Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis. PMID:26339978

ELISA microarray technology as a high-throughput system for cancer biomarker validation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zangar, Richard C.; Daly, Don S.; White, Amanda M.

A large gap currently exists between the ability to discover potential biomarkers and the ability to assess the real value of these proteins for cancer screening. One major challenge in biomarker validation is the inherent variability in biomarker levels. This variability stems from the diversity across the human population and the considerable molecular heterogeneity between individual tumors, even those that originate from a single tissue. Another major challenge with cancer screening is that most cancers are rare in the general population, meaning that the specificity of an assay must be very high if the number of false positive is notmore » going to be much greater than the number of true positives. Because of these challenges with biomarker validation, it is necessary to analysis of thousands of samples before a clear idea of the utility of a screening assay can be determined. Enzyme-linked immunosorbent assay (ELISA) microarray technology can simultaneously quantify levels of multiple proteins and has the potential to accelerate biomarker validation. In this review, we discuss current ELISA microarray technology and the enabling advances needed to achieve the reproducibility and throughput that are required to evaluate cancer biomarkers.« less

mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.

PubMed

Scheidl, Stefan J; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

2002-03-01

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

Recursive feature selection with significant variables of support vectors.

PubMed

Tsai, Chen-An; Huang, Chien-Hsun; Chang, Ching-Wei; Chen, Chun-Houh

2012-01-01

The development of DNA microarray makes researchers screen thousands of genes simultaneously and it also helps determine high- and low-expression level genes in normal and disease tissues. Selecting relevant genes for cancer classification is an important issue. Most of the gene selection methods use univariate ranking criteria and arbitrarily choose a threshold to choose genes. However, the parameter setting may not be compatible to the selected classification algorithms. In this paper, we propose a new gene selection method (SVM-t) based on the use of t-statistics embedded in support vector machine. We compared the performance to two similar SVM-based methods: SVM recursive feature elimination (SVMRFE) and recursive support vector machine (RSVM). The three methods were compared based on extensive simulation experiments and analyses of two published microarray datasets. In the simulation experiments, we found that the proposed method is more robust in selecting informative genes than SVMRFE and RSVM and capable to attain good classification performance when the variations of informative and noninformative genes are different. In the analysis of two microarray datasets, the proposed method yields better performance in identifying fewer genes with good prediction accuracy, compared to SVMRFE and RSVM.

Transcriptional profiling of the parr–smolt transformation in Atlantic salmon

USGS Publications Warehouse

Robertson, Laura S.; McCormick, Stephen D.

2012-01-01

The parr–smolt transformation in Atlantic salmon (Salmo salar) is a complex developmental process that culminates in the ability to migrate to and live in seawater. We used GRASP 16K cDNA microarrays to identify genes that are differentially expressed in the liver, gill, hypothalamus, pituitary, and olfactory rosettes of smolts compared to parr. Smolts had higher levels of gill Na+/K+-ATPase activity, plasma cortisol and plasma thyroid hormones relative to parr. Across all five tissues, stringent microarray analyses identified 48 features that were differentially expressed in smolts compared to parr. Using a less stringent method we found 477 features that were differentially expressed at least 1.2-fold in smolts, including 172 features in the gill. Smolts had higher mRNA levels of genes involved in transcription, protein biosynthesis and folding, electron transport, oxygen transport, and sensory perception and lower mRNA levels for genes involved in proteolysis. Quantitative RT-PCR was used to confirm differential expression in select genes identified by microarray analyses and to quantify expression of other genes known to be involved in smolting. This study expands our understanding of the molecular processes that underlie smolting in Atlantic salmon and identifies genes for further investigation.

The oncogenic gene fusion TMPRSS2: ERG is not a diagnostic or prognostic marker for ovarian cancer

PubMed Central

Huang, Lillian; Schauer, Isaiah G; Zhang, Jing; Mercado-Uribe, Imelda; Deavers, Michael T; Huang, Jiaoti; Liu, Jinsong

2011-01-01

TMPRSS2:ERG is a gene fusion resulting from the chromosomal rearrangement of the androgen-regulated TMPRSS2 gene and the ETS transcription factor ERG, leading to the over-expression of the oncogenic molecule ERG. This gene rearrangement has been found in approximately half of all prostate cancers and ERG overexpression is considered as a novel diagnostic marker for prostate carcinoma. However, little is known about the role of the TMPRSS2:ERG gene fusion in ovarian cancer. The purpose of this study was to test ERG expression in ovarian cancer and its potential as a diagnostic marker for ovarian carcinoma progression. A tissue microarray containing 180 ovarian cancer tissues of various pathological types and grades were examined by immunohistochemical analysis for expression of ERG. We also used 40 prostate carcinoma tissues and 40 normal tissues for comparison in parallel experiments. ERG-positive expression was detected in 40% of the prostate tumor cancer, as well as in internal positive control endothelial cells, confirming over-expression of ERG in prostate cancer at relatively the same rate observed by others. In contrast, all of the ovarian tumor patient tissues of varying histologic types were ERG-negative, despite some positivity in endothelial cells. These results suggest that the oncogenic gene fusion TMPRSS2:ERG does not occur in ovarian cancer relative to prostate cancer. Therefore, development of ERG expression profile would not be a useful diagnostic or prognostic marker for ovarian cancer patient screening. PMID:22076164

The oncogenic gene fusion TMPRSS2: ERG is not a diagnostic or prognostic marker for ovarian cancer.

PubMed

Huang, Lillian; Schauer, Isaiah G; Zhang, Jing; Mercado-Uribe, Imelda; Deavers, Michael T; Huang, Jiaoti; Liu, Jinsong

2011-01-01

TMPRSS2:ERG is a gene fusion resulting from the chromosomal rearrangement of the androgen-regulated TMPRSS2 gene and the ETS transcription factor ERG, leading to the over-expression of the oncogenic molecule ERG. This gene rearrangement has been found in approximately half of all prostate cancers and ERG overexpression is considered as a novel diagnostic marker for prostate carcinoma. However, little is known about the role of the TMPRSS2:ERG gene fusion in ovarian cancer. The purpose of this study was to test ERG expression in ovarian cancer and its potential as a diagnostic marker for ovarian carcinoma progression. A tissue microarray containing 180 ovarian cancer tissues of various pathological types and grades were examined by immunohistochemical analysis for expression of ERG. We also used 40 prostate carcinoma tissues and 40 normal tissues for comparison in parallel experiments. ERG-positive expression was detected in 40% of the prostate tumor cancer, as well as in internal positive control endothelial cells, confirming over-expression of ERG in prostate cancer at relatively the same rate observed by others. In contrast, all of the ovarian tumor patient tissues of varying histologic types were ERG-negative, despite some positivity in endothelial cells. These results suggest that the oncogenic gene fusion TMPRSS2:ERG does not occur in ovarian cancer relative to prostate cancer. Therefore, development of ERG expression profile would not be a useful diagnostic or prognostic marker for ovarian cancer patient screening.

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm.

PubMed

Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

2015-01-01

DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

PubMed

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong; Li, Ning

2016-12-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma

PubMed Central

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong

2016-01-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC (n = 65) and healthy control subjects (n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC. PMID:27885040

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.

PubMed

Tramontana, S; Bionaz, M; Sharma, A; Graugnard, D E; Cutler, E A; Ajmone-Marsan, P; Hurley, W L; Loor, J J

2008-08-01

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species.