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

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

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.

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

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.

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.

Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium

PubMed Central

Howat, William J; Blows, Fiona M; Provenzano, Elena; Brook, Mark N; Morris, Lorna; Gazinska, Patrycja; Johnson, Nicola; McDuffus, Leigh‐Anne; Miller, Jodi; Sawyer, Elinor J; Pinder, Sarah; van Deurzen, Carolien H M; Jones, Louise; Sironen, Reijo; Visscher, Daniel; Caldas, Carlos; Daley, Frances; Coulson, Penny; Broeks, Annegien; Sanders, Joyce; Wesseling, Jelle; Nevanlinna, Heli; Fagerholm, Rainer; Blomqvist, Carl; Heikkilä, Päivi; Ali, H Raza; Dawson, Sarah‐Jane; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli‐Matti; Cox, Angela; Brock, Ian W; Cross, Simon S; Reed, Malcolm W; Couch, Fergus J; Olson, Janet E; Devillee, Peter; Mesker, Wilma E; Seyaneve, Caroline M; Hollestelle, Antoinette; Benitez, Javier; Perez, Jose Ignacio Arias; Menéndez, Primitiva; Bolla, Manjeet K; Easton, Douglas F; Schmidt, Marjanka K; Pharoah, Paul D; Sherman, Mark E

2014-01-01

Abstract Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large‐scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65–70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose‐response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96–98%), but yielded many false positives (positive predictive value = 30–32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large‐scale, quantitative scoring of immunohistochemically stained tissue microarrays available in consortia. However, continued optimization, rigorous marker‐specific quality control measures and standardization of tissue microarray designs, staining and scoring protocols is needed to enhance results. PMID:27499890

FlyAtlas 2: a new version of the Drosophila melanogaster expression atlas with RNA-Seq, miRNA-Seq and sex-specific data

PubMed Central

Krause, Sue A; Pandit, Aniruddha; Davies, Shireen A

2018-01-01

Abstract FlyAtlas 2 (www.flyatlas2.org) is part successor, part complement to the FlyAtlas database and web application for studying the expression of the genes of Drosophila melanogaster in different tissues of adults and larvae. Although generated in the same lab with the same fly line raised on the same diet as FlyAtlas, the FlyAtlas2 resource employs a completely new set of expression data based on RNA-Seq, rather than microarray analysis, and so it allows the user to obtain information for the expression of different transcripts of a gene. Furthermore, the data for somatic tissues are now available for both male and female adult flies, allowing studies of sexual dimorphism. Gene coverage has been extended by the inclusion of microRNAs and many of the RNA genes included in Release 6 of the Drosophila reference genome. The web interface has been modified to accommodate the extra data, but at the same time has been adapted for viewing on small mobile devices. Users also have access to the RNA-Seq reads displayed alongside the annotated Drosophila genome in the (external) UCSC browser, and are able to link out to the previous FlyAtlas resource to compare the data obtained by RNA-Seq with that obtained using microarrays. PMID:29069479

An informatics model for tissue banks--lessons learned from the Cooperative Prostate Cancer Tissue Resource.

PubMed

Patel, Ashokkumar A; Gilbertson, John R; Parwani, Anil V; Dhir, Rajiv; Datta, Milton W; Gupta, Rajnish; Berman, Jules J; Melamed, Jonathan; Kajdacsy-Balla, Andre; Orenstein, Jan; Becich, Michael J

2006-05-05

Advances in molecular biology and growing requirements from biomarker validation studies have generated a need for tissue banks to provide quality-controlled tissue samples with standardized clinical annotation. The NCI Cooperative Prostate Cancer Tissue Resource (CPCTR) is a distributed tissue bank that comprises four academic centers and provides thousands of clinically annotated prostate cancer specimens to researchers. Here we describe the CPCTR information management system architecture, common data element (CDE) development, query interfaces, data curation, and quality control. Data managers review the medical records to collect and continuously update information for the 145 clinical, pathological and inventorial CDEs that the Resource maintains for each case. An Access-based data entry tool provides de-identification and a standard communication mechanism between each group and a central CPCTR database. Standardized automated quality control audits have been implemented. Centrally, an Oracle database has web interfaces allowing multiple user-types, including the general public, to mine de-identified information from all of the sites with three levels of specificity and granularity as well as to request tissues through a formal letter of intent. Since July 2003, CPCTR has offered over 6,000 cases (38,000 blocks) of highly characterized prostate cancer biospecimens, including several tissue microarrays (TMA). The Resource developed a website with interfaces for the general public as well as researchers and internal members. These user groups have utilized the web-tools for public query of summary data on the cases that were available, to prepare requests, and to receive tissues. As of December 2005, the Resource received over 130 tissue requests, of which 45 have been reviewed, approved and filled. Additionally, the Resource implemented the TMA Data Exchange Specification in its TMA program and created a computer program for calculating PSA recurrence. Building a biorepository infrastructure that meets today's research needs involves time and input of many individuals from diverse disciplines. The CPCTR can provide large volumes of carefully annotated prostate tissue for research initiatives such as Specialized Programs of Research Excellence (SPOREs) and for biomarker validation studies and its experience can help development of collaborative, large scale, virtual tissue banks in other organ systems.

The AIDS and Cancer Specimen Resource: Role in HIV/AIDS scientific discovery

PubMed Central

Ayers, Leona W; Silver, Sylvia; McGrath, Michael S; Orenstein, Jan M

2007-01-01

The AIDS Cancer and Specimen Resource (ACSR) supports scientific discovery in the area of HIV/AIDS-associated malignancies. The ACSR was established as a cooperative agreement between the NCI (Office of the Director, Division of Cancer Treatment and Diagnosis) and regional consortia, University of California, San Francisco (West Coast), George Washington University (East Coast) and Ohio State University (Mid-Region) to collect, preserve and disperse HIV-related tissues and biologic fluids and controls along with clinical data to qualified investigators. The available biological samples with clinical data and the application process are described on the ACSR web site. The ACSR tissue bank has more than 100,000 human HIV positive specimens that represent different processing (43), specimen (15), and anatomical site (50) types. The ACSR provides special biospecimen collections and prepares speciality items, e.g., tissue microarrays (TMA), DNA libraries. Requests have been greatest for Kaposi's sarcoma (32%) and non-Hodgkin's lymphoma (26%). Dispersed requests include 83% tissue (frozen and paraffin embedded), 18% plasma/serum and 9% other. ACSR also provides tissue microarrays of, e.g., Kaposi's sarcoma and non-Hodgkin's lymphoma, for biomarker assays and has developed collaborations with other groups that provide access to additional AIDS-related malignancy specimens. ACSR members and associates have completed 63 podium and poster presentations. Investigators have submitted 125 letters of intent requests. Discoveries using ACSR have been reported in 61 scientific publications in notable journals with an average impact factor of 7. The ACSR promotes the scientific exploration of the relationship between HIV/AIDS and malignancy by participation at national and international scientific meetings, contact with investigators who have productive research in this area and identifying, collecting, preserving, enhancing, and dispersing HIV/AIDS-related malignancy specimens to funded, approved researchers at no fee. Scientific discovery has been advanced by this unique biorepository. Investigators are encouraged to browse the ACSR Internet site for materials to enhance their own scientific initiatives. PMID:17335575

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.

National Mesothelioma Virtual Bank: A Platform for Collaborative Research and Mesothelioma Biobanking Resource to Support Translational Research

PubMed Central

Parwani, Anil V.; Melamed, Jonathan; Flores, Raja; Pennathur, Arjun; Valdivieso, Federico; Whelan, Nancy B.; Landreneau, Rodeny; Luketich, James; Feldman, Michael; Pass, Harvey I.; Becich, Michael J.

2013-01-01

The National Mesothelioma Virtual Bank (NMVB), developed six years ago, gathers clinically annotated human mesothelioma specimens for basic and clinical science research. During this period, this resource has greatly increased its collection of specimens by expanding the number of contributing academic health centers including New York University, University of Pennsylvania, University of Pittsburgh Medical Center, and Mount Sinai School of Medicine. Marketing efforts at both national and international annual conferences increase awareness and availability of the mesothelioma specimens at no cost to approved investigators, who query the web-based NMVB database for cumulative and appropriate patient clinicopathological information on the specimens. The data disclosure and specimen distribution protocols are tightly regulated to maintain compliance with participating institutions' IRB and regulatory committee reviews. The NMVB currently has over 1120 annotated cases available for researchers, including paraffin embedded tissues, fresh frozen tissue, tissue microarrays (TMA), blood samples, and genomic DNA. In addition, the resource offers expertise and assistance for collaborative research. Furthermore, in the last six years, the resource has provided hundreds of specimens to the research community. The investigators can request specimens and/or data by submitting a Letter of Intent (LOI) that is evaluated by NMVB research evaluation panel (REP). PMID:26316942

National Mesothelioma Virtual Bank: A Platform for Collaborative Research and Mesothelioma Biobanking Resource to Support Translational Research.

PubMed

Amin, Waqas; Parwani, Anil V; Melamed, Jonathan; Flores, Raja; Pennathur, Arjun; Valdivieso, Federico; Whelan, Nancy B; Landreneau, Rodeny; Luketich, James; Feldman, Michael; Pass, Harvey I; Becich, Michael J

2013-01-01

The National Mesothelioma Virtual Bank (NMVB), developed six years ago, gathers clinically annotated human mesothelioma specimens for basic and clinical science research. During this period, this resource has greatly increased its collection of specimens by expanding the number of contributing academic health centers including New York University, University of Pennsylvania, University of Pittsburgh Medical Center, and Mount Sinai School of Medicine. Marketing efforts at both national and international annual conferences increase awareness and availability of the mesothelioma specimens at no cost to approved investigators, who query the web-based NMVB database for cumulative and appropriate patient clinicopathological information on the specimens. The data disclosure and specimen distribution protocols are tightly regulated to maintain compliance with participating institutions' IRB and regulatory committee reviews. The NMVB currently has over 1120 annotated cases available for researchers, including paraffin embedded tissues, fresh frozen tissue, tissue microarrays (TMA), blood samples, and genomic DNA. In addition, the resource offers expertise and assistance for collaborative research. Furthermore, in the last six years, the resource has provided hundreds of specimens to the research community. The investigators can request specimens and/or data by submitting a Letter of Intent (LOI) that is evaluated by NMVB research evaluation panel (REP).

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

THE ABRF MARG MICROARRAY SURVEY 2005: TAKING THE PULSE ON THE MICROARRAY FIELD

EPA Science Inventory

Over the past several years microarray technology has evolved into a critical component of any discovery based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a pr...

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.

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

Tissues from population-based cancer registries: a novel approach to increasing research potential.

PubMed

Goodman, Marc T; Hernandez, Brenda Y; Hewitt, Stephen; Lynch, Charles F; Coté, Timothy R; Frierson, Henry F; Moskaluk, Christopher A; Killeen, Jeffrey L; Cozen, Wendy; Key, Charles R; Clegg, Limin; Reichman, Marsha; Hankey, Benjamin F; Edwards, Brenda

2005-07-01

Population-based cancer registries, such as those included in the Surveillance, Epidemiology, and End-Results (SEER) Program, offer tremendous research potential beyond traditional surveillance activities. We describe the expansion of SEER registries to gather formalin-fixed, paraffin-embedded tissue from cancer patients on a population basis. Population-based tissue banks have the advantage of providing an unbiased sampling frame for evaluating the public health impact of genes or protein targets that may be used for therapeutic or diagnostic purposes in defined communities. Such repositories provide a unique resource for testing new molecular classification schemes for cancer, validating new biologic markers of malignancy, prognosis and progression, assessing therapeutic targets, and measuring allele frequencies of cancer-associated genetic polymorphisms or germline mutations in representative samples. The assembly of tissue microarrays will allow for the use of rapid, large-scale protein-expression profiling of tumor samples while limiting depletion of this valuable resource. Access to biologic specimens through SEER registries will provide researchers with demographic, clinical, and risk factor information on cancer patients with assured data quality and completeness. Clinical outcome data, such as disease-free survival, can be correlated with previously validated prognostic markers. Furthermore, the anonymity of the study subject can be protected through rigorous standards of confidentiality. SEER-based tissue resources represent a step forward in true, population-based tissue repositories of tumors from US patients and may serve as a foundation for molecular epidemiology studies of cancer in this country.

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.

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.

Cell and tissue microarray technologies for protein and nucleic acid expression profiling.

PubMed

Cardano, Marina; Diaferia, Giuseppe R; Falavigna, Maurizio; Spinelli, Chiara C; Sessa, Fausto; DeBlasio, Pasquale; Biunno, Ida

2013-02-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

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

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

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

Deep learning for tissue microarray image-based outcome prediction in patients with colorectal cancer

NASA Astrophysics Data System (ADS)

Bychkov, Dmitrii; Turkki, Riku; Haglund, Caj; Linder, Nina; Lundin, Johan

2016-03-01

Recent advances in computer vision enable increasingly accurate automated pattern classification. In the current study we evaluate whether a convolutional neural network (CNN) can be trained to predict disease outcome in patients with colorectal cancer based on images of tumor tissue microarray samples. We compare the prognostic accuracy of CNN features extracted from the whole, unsegmented tissue microarray spot image, with that of CNN features extracted from the epithelial and non-epithelial compartments, respectively. The prognostic accuracy of visually assessed histologic grade is used as a reference. The image data set consists of digitized hematoxylin-eosin (H and E) stained tissue microarray samples obtained from 180 patients with colorectal cancer. The patient samples represent a variety of histological grades, have data available on a series of clinicopathological variables including long-term outcome and ground truth annotations performed by experts. The CNN features extracted from images of the epithelial tissue compartment significantly predicted outcome (hazard ratio (HR) 2.08; CI95% 1.04-4.16; area under the curve (AUC) 0.66) in a test set of 60 patients, as compared to the CNN features extracted from unsegmented images (HR 1.67; CI95% 0.84-3.31, AUC 0.57) and visually assessed histologic grade (HR 1.96; CI95% 0.99-3.88, AUC 0.61). As a conclusion, a deep-learning classifier can be trained to predict outcome of colorectal cancer based on images of H and E stained tissue microarray samples and the CNN features extracted from the epithelial compartment only resulted in a prognostic discrimination comparable to that of visually determined histologic grade.

Proteomic analysis of formalin-fixed paraffin embedded tissue by MALDI imaging mass spectrometry

PubMed Central

Casadonte, Rita; Caprioli, Richard M

2012-01-01

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

Research resource: Tissue-specific transcriptomics and cistromics of nuclear receptor signaling: a web research resource.

PubMed

Ochsner, Scott A; Watkins, Christopher M; LaGrone, Benjamin S; Steffen, David L; McKenna, Neil J

2010-10-01

Nuclear receptors (NRs) are ligand-regulated transcription factors that recruit coregulators and other transcription factors to gene promoters to effect regulation of tissue-specific transcriptomes. The prodigious rate at which the NR signaling field has generated high content gene expression and, more recently, genome-wide location analysis datasets has not been matched by a committed effort to archiving this information for routine access by bench and clinical scientists. As a first step towards this goal, we searched the MEDLINE database for studies, which referenced either expression microarray and/or genome-wide location analysis datasets in which a NR or NR ligand was an experimental variable. A total of 1122 studies encompassing 325 unique organs, tissues, primary cells, and cell lines, 35 NRs, and 91 NR ligands were retrieved and annotated. The data were incorporated into a new section of the Nuclear Receptor Signaling Atlas Molecule Pages, Transcriptomics and Cistromics, for which we designed an intuitive, freely accessible user interface to browse the studies. Each study links to an abstract, the MEDLINE record, and, where available, Gene Expression Omnibus and ArrayExpress records. The resource will be updated on a regular basis to provide a current and comprehensive entrez into the sum of transcriptomic and cistromic research in this field.

Recurrent pregnancy loss evaluation combined with 24-chromosome microarray of miscarriage tissue provides a probable or definite cause of pregnancy loss in over 90% of patients.

PubMed

Popescu, F; Jaslow, C R; Kutteh, W H

2018-04-01

Will the addition of 24-chromosome microarray analysis on miscarriage tissue combined with the standard American Society for Reproductive Medicine (ASRM) evaluation for recurrent miscarriage explain most losses? Over 90% of patients with recurrent pregnancy loss (RPL) will have a probable or definitive cause identified when combining genetic testing on miscarriage tissue with the standard ASRM evaluation for recurrent miscarriage. RPL is estimated to occur in 2-4% of reproductive age couples. A probable cause can be identified in approximately 50% of patients after an ASRM recommended workup including an evaluation for parental chromosomal abnormalities, congenital and acquired uterine anomalies, endocrine imbalances and autoimmune factors including antiphospholipid syndrome. Single-center, prospective cohort study that included 100 patients seen in a private RPL clinic from 2014 to 2017. All 100 women had two or more pregnancy losses, a complete evaluation for RPL as defined by the ASRM, and miscarriage tissue evaluated by 24-chromosome microarray analysis after their second or subsequent miscarriage. Frequencies of abnormal results for evidence-based diagnostic tests considered definite or probable causes of RPL (karyotyping for parental chromosomal abnormalities, and 24-chromosome microarray evaluation for products of conception (POC); pelvic sonohysterography, hysterosalpingogram, or hysteroscopy for uterine anomalies; immunological tests for lupus anticoagulant and anticardiolipin antibodies; and blood tests for thyroid stimulating hormone (TSH), prolactin and hemoglobin A1c) were evaluated. We excluded cases where there was maternal cell contamination of the miscarriage tissue or if the ASRM evaluation was incomplete. A cost analysis for the evaluation of RPL was conducted to determine whether a proposed procedure of 24-chromome microarray evaluation followed by an ASRM RPL workup (for those RPL patients who had a normal 24-chromosome microarray evaluation) was more cost-efficient than conducting ASRM RPL workups on RPL patients followed by 24-chromosome microarray analysis (for those RPL patients who had a normal RPL workup). A definite or probable cause of pregnancy loss was identified in the vast majority (95/100; 95%) of RPL patients when a 24-chromosome pair microarray evaluation of POC testing is combined with the standard ASRM RPL workup evaluation at the time of the second or subsequent loss. The ASRM RPL workup identified an abnormality and a probable explanation for pregnancy loss in only 45/100 or 45% of all patients. A definite abnormality was identified in 67/100 patients or 67% when initial testing was performed using 24-chromosome microarray analyses on the miscarriage tissue. Only 5/100 (5%) patients, who had a euploid loss and a normal ASRM RPL workup, had a pregnancy loss without a probable or definitive cause identified. All other losses were explained by an abnormal 24-chromosome microarray analysis of the miscarriage tissue, an abnormal finding of the RPL workup, or a combination of both. Results from the cost analysis indicated that an initial approach of using a 24-chromosome microarray analysis on miscarriage tissue resulted in a 50% savings in cost to the health care system and to the patient. This is a single-center study on a small group of well-characterized women with RPL. There was an incomplete follow-up on subsequent pregnancy outcomes after evaluation, however this should not affect our principal results. The maternal age of patients varied from 26 to 45 years old. More aneuploid pregnancy losses would be expected in older women, particularly over the age of 35 years old. Evaluation of POC using 24-chromosome microarray analysis adds significantly to the ASRM recommended evaluation of RPL. Genetic evaluation on miscarriage tissue obtained at the time of the second and subsequent pregnancy losses should be offered to all couples with two or more consecutive pregnancy losses. The combination of a genetic evaluation on miscarriage tissue with an evidence-based evaluation for RPL will identify a probable or definitive cause in over 90% of miscarriages. No funding was received for this study and there are no conflicts of interest to declare. Not applicable.

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.

Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling

PubMed Central

Cardano, Marina; Diaferia, Giuseppe R.; Falavigna, Maurizio; Spinelli, Chiara C.; Sessa, Fausto; DeBlasio, Pasquale

2013-01-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform. PMID:23172795

Evaluation of two outlier-detection-based methods for detecting tissue-selective genes from microarray data.

PubMed

Kadota, Koji; Konishi, Tomokazu; Shimizu, Kentaro

2007-05-01

Large-scale expression profiling using DNA microarrays enables identification of tissue-selective genes for which expression is considerably higher and/or lower in some tissues than in others. Among numerous possible methods, only two outlier-detection-based methods (an AIC-based method and Sprent's non-parametric method) can treat equally various types of selective patterns, but they produce substantially different results. We investigated the performance of these two methods for different parameter settings and for a reduced number of samples. We focused on their ability to detect selective expression patterns robustly. We applied them to public microarray data collected from 36 normal human tissue samples and analyzed the effects of both changing the parameter settings and reducing the number of samples. The AIC-based method was more robust in both cases. The findings confirm that the use of the AIC-based method in the recently proposed ROKU method for detecting tissue-selective expression patterns is correct and that Sprent's method is not suitable for ROKU.

Microarrays in brain research: the good, the bad and the ugly.

PubMed

Mirnics, K

2001-06-01

Making sense of microarray data is a complex process, in which the interpretation of findings will depend on the overall experimental design and judgement of the investigator performing the analysis. As a result, differences in tissue harvesting, microarray types, sample labelling and data analysis procedures make post hoc sharing of microarray data a great challenge. To ensure rapid and meaningful data exchange, we need to create some order out of the existing chaos. In these ground-breaking microarray standardization and data sharing efforts, NIH agencies should take a leading role

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

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

Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection inmore » archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.« less

Manufacturing of microarrays.

PubMed

Petersen, David W; Kawasaki, Ernest S

2007-01-01

DNA microarray technology has become a powerful tool in the arsenal of the molecular biologist. Capitalizing on high precision robotics and the wealth of DNA sequences annotated from the genomes of a large number of organisms, the manufacture of microarrays is now possible for the average academic laboratory with the funds and motivation. Microarray production requires attention to both biological and physical resources, including DNA libraries, robotics, and qualified personnel. While the fabrication of microarrays is a very labor-intensive process, production of quality microarrays individually tailored on a project-by-project basis will help researchers shed light on future scientific questions.

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

EMAAS: An extensible grid-based Rich Internet Application for microarray data analysis and management

PubMed Central

Barton, G; Abbott, J; Chiba, N; Huang, DW; Huang, Y; Krznaric, M; Mack-Smith, J; Saleem, A; Sherman, BT; Tiwari, B; Tomlinson, C; Aitman, T; Darlington, J; Game, L; Sternberg, MJE; Butcher, SA

2008-01-01

Background Microarray experimentation requires the application of complex analysis methods as well as the use of non-trivial computer technologies to manage the resultant large data sets. This, together with the proliferation of tools and techniques for microarray data analysis, makes it very challenging for a laboratory scientist to keep up-to-date with the latest developments in this field. Our aim was to develop a distributed e-support system for microarray data analysis and management. Results EMAAS (Extensible MicroArray Analysis System) is a multi-user rich internet application (RIA) providing simple, robust access to up-to-date resources for microarray data storage and analysis, combined with integrated tools to optimise real time user support and training. The system leverages the power of distributed computing to perform microarray analyses, and provides seamless access to resources located at various remote facilities. The EMAAS framework allows users to import microarray data from several sources to an underlying database, to pre-process, quality assess and analyse the data, to perform functional analyses, and to track data analysis steps, all through a single easy to use web portal. This interface offers distance support to users both in the form of video tutorials and via live screen feeds using the web conferencing tool EVO. A number of analysis packages, including R-Bioconductor and Affymetrix Power Tools have been integrated on the server side and are available programmatically through the Postgres-PLR library or on grid compute clusters. Integrated distributed resources include the functional annotation tool DAVID, GeneCards and the microarray data repositories GEO, CELSIUS and MiMiR. EMAAS currently supports analysis of Affymetrix 3' and Exon expression arrays, and the system is extensible to cater for other microarray and transcriptomic platforms. Conclusion EMAAS enables users to track and perform microarray data management and analysis tasks through a single easy-to-use web application. The system architecture is flexible and scalable to allow new array types, analysis algorithms and tools to be added with relative ease and to cope with large increases in data volume. PMID:19032776

Evaluation of Two Outlier-Detection-Based Methods for Detecting Tissue-Selective Genes from Microarray Data

PubMed Central

Kadota, Koji; Konishi, Tomokazu; Shimizu, Kentaro

2007-01-01

Large-scale expression profiling using DNA microarrays enables identification of tissue-selective genes for which expression is considerably higher and/or lower in some tissues than in others. Among numerous possible methods, only two outlier-detection-based methods (an AIC-based method and Sprent’s non-parametric method) can treat equally various types of selective patterns, but they produce substantially different results. We investigated the performance of these two methods for different parameter settings and for a reduced number of samples. We focused on their ability to detect selective expression patterns robustly. We applied them to public microarray data collected from 36 normal human tissue samples and analyzed the effects of both changing the parameter settings and reducing the number of samples. The AIC-based method was more robust in both cases. The findings confirm that the use of the AIC-based method in the recently proposed ROKU method for detecting tissue-selective expression patterns is correct and that Sprent’s method is not suitable for ROKU. PMID:19936074

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.

Interlaboratory comparison of immunohistochemical testing for HER2: results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey.

PubMed

Fitzgibbons, Patrick L; Murphy, Douglas A; Dorfman, David M; Roche, Patrick C; Tubbs, Raymond R

2006-10-01

Correct assessment of human epidermal growth factor receptor 2 (HER2) status is essential in managing patients with invasive breast carcinoma, but few data are available on the accuracy of laboratories performing HER2 testing by immunohistochemistry (IHC). To review the results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey. The HER2 survey is designed for laboratories performing immunohistochemical staining and interpretation for HER2. The survey uses tissue microarrays, each consisting of ten 3-mm tissue cores obtained from different invasive breast carcinomas. All cases are also analyzed by fluorescence in situ hybridization. Participants receive 8 tissue microarrays (80 cases) with instructions to perform immunostaining for HER2 using the laboratory's standard procedures. The laboratory interprets the stained slides and returns results to the College of American Pathologists for analysis. In 2004 and 2005, a core was considered "graded" when at least 90% of laboratories agreed on the result--negative (0, 1+) versus positive (2+, 3+). This interlaboratory comparison survey included 102 laboratories in 2004 and 141 laboratories in 2005. Of the 160 cases in both surveys, 111 (69%) achieved 90% consensus (graded). All 43 graded cores scored as IHC-positive were fluorescence in situ hybridization-positive, whereas all but 3 of the 68 IHC-negative graded cores were fluorescence in situ hybridization-negative. Ninety-seven (95%) of 102 laboratories in 2004 and 129 (91%) of 141 laboratories in 2005 correctly scored at least 90% of the graded cores. Performance among laboratories performing HER2 IHC in this tissue microarray-based survey was excellent. Cores found to be IHC-positive or IHC-negative by participant consensus can be used as validated benchmarks for interlaboratory comparison, allowing laboratories to assess their performance and determine if improvements are needed.

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

Recovering from iron deficiency chlorosis in near-isogenic soybeans: a microarray study.

PubMed

O'Rourke, Jamie A; Graham, Michelle A; Vodkin, Lila; Gonzalez, Delkin Orlando; Cianzio, Silvia R; Shoemaker, Randy C

2007-05-01

Iron deficiency chlorosis (IDC) in soybeans has proven to be a perennial problem in the calcareous soils of the U.S. upper Midwest. A historically difficult trait to study in fields, the use of hydroponics in a controlled greenhouse environment has provided a mechanism to study genetic variation while limiting environmental complications. IDC susceptible plants growing in calcareous soils and in iron-controlled hydroponic experiments often exhibit a characteristic chlorotic phenotype early in the growing season but are able to re-green later in the season. To examine the changes in gene expression of these plants, near-isogenic lines, iron efficient PI548553 (Clark) and iron inefficient PI547430 (IsoClark), developed for their response to iron deficiency stress [USDA, ARS, National Genetic Resources Program, Germplasm Resources Information Network - GRIN. (Online Database) National Germplasm Resources Laboratory, Beltsville, MD, 2004. Available: http://www.ars.grin.gov/cgi-bin/npgs/html/acc_search.pl?accid=PI+547430. [22] were grown in iron-deficient hydroponic conditions for one week, then transferred to iron sufficient conditions for another week. This induced a phenotypic response mimicking the growth of the plants in the field; initial chlorosis followed by re-greening. RNA was isolated from root tissue and transcript profiles were examined between the two near-isogenic lines using publicly available cDNA microarrays. By alleviating the iron deficiency stress our expectation was that plants would return to baseline expression levels. However, the microarray comparison identified four cDNAs that were under-expressed by a two-fold or greater difference in the iron inefficient plant compared to the iron efficient plant. This differential expression was re-examined and confirmed by real time PCR experimentation. Control experiments showed that these genes are not differentially expressed in plants grown continually under iron rich hydroponic conditions. The expression differences suggest potential residual effects of iron deficiency on plant health.

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

PubMed Central

Ramsey, John S; Wilson, Alex CC; de Vos, Martin; Sun, Qi; Tamborindeguy, Cecilia; Winfield, Agnese; Malloch, Gaynor; Smith, Dawn M; Fenton, Brian; Gray, Stewart M; Jander, Georg

2007-01-01

Background The green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. Results Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. Conclusion New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects. PMID:18021414

Genomic analysis of Fusarium verticillioides.

PubMed

Brown, D W; Butchko, R A E; Proctor, R H

2008-09-01

Fusarium verticillioides (teleomorph Gibberella moniliformis) can be either an endophyte of maize, causing no visible disease, or a pathogen-causing disease of ears, stalks, roots and seedlings. At any stage, this fungus can synthesize fumonisins, a family of mycotoxins structurally similar to the sphingolipid sphinganine. Ingestion of fumonisin-contaminated maize has been associated with a number of animal diseases, including cancer in rodents, and exposure has been correlated with human oesophageal cancer in some regions of the world, and some evidence suggests that fumonisins are a risk factor for neural tube defects. A primary goal of the authors' laboratory is to eliminate fumonisin contamination of maize and maize products. Understanding how and why these toxins are made and the F. verticillioides-maize disease process will allow one to develop novel strategies to limit tissue destruction (rot) and fumonisin production. To meet this goal, genomic sequence data, expressed sequence tags (ESTs) and microarrays are being used to identify F. verticillioides genes involved in the biosynthesis of toxins and plant pathogenesis. This paper describes the current status of F. verticillioides genomic resources and three approaches being used to mine microarray data from a wild-type strain cultured in liquid fumonisin production medium for 12, 24, 48, 72, 96 and 120h. Taken together, these approaches demonstrate the power of microarray technology to provide information on different biological processes.

The role of metalloendopeptidases in oropharyngeal carcinomas assessed by tissue microarray.

PubMed

Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Noguti, Juliana; Oshima, Celina T F; Ihara, Silvia S M; Franco, Marcello F

2011-01-01

The goal of this study was to investigate the expression of some metalloendopeptidases in squamous cell carcinomas of the oropharynx as well as its 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 EP24.15 and EP24.16 by means of tissue microarrays. Expression of EP24.15 or EP24.16 was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinomas of the oropharynx. In summary, our results support the notion that EP24.15 and EP24.16 are expressed in carcinoma of the oropharynx; however, these do not appear to be suitable biomarkers for histological grading, disease stage or recurrence as depicted by tissue microarrays and immunohistochemistry.

The PEPR GeneChip data warehouse, and implementation of a dynamic time series query tool (SGQT) with graphical interface.

PubMed

Chen, Josephine; Zhao, Po; Massaro, Donald; Clerch, Linda B; Almon, Richard R; DuBois, Debra C; Jusko, William J; Hoffman, Eric P

2004-01-01

Publicly accessible DNA databases (genome browsers) are rapidly accelerating post-genomic research (see http://www.genome.ucsc.edu/), with integrated genomic DNA, gene structure, EST/ splicing and cross-species ortholog data. DNA databases have relatively low dimensionality; the genome is a linear code that anchors all associated data. In contrast, RNA expression and protein databases need to be able to handle very high dimensional data, with time, tissue, cell type and genes, as interrelated variables. The high dimensionality of microarray expression profile data, and the lack of a standard experimental platform have complicated the development of web-accessible databases and analytical tools. We have designed and implemented a public resource of expression profile data containing 1024 human, mouse and rat Affymetrix GeneChip expression profiles, generated in the same laboratory, and subject to the same quality and procedural controls (Public Expression Profiling Resource; PEPR). Our Oracle-based PEPR data warehouse includes a novel time series query analysis tool (SGQT), enabling dynamic generation of graphs and spreadsheets showing the action of any transcript of interest over time. In this report, we demonstrate the utility of this tool using a 27 time point, in vivo muscle regeneration series. This data warehouse and associated analysis tools provides access to multidimensional microarray data through web-based interfaces, both for download of all types of raw data for independent analysis, and also for straightforward gene-based queries. Planned implementations of PEPR will include web-based remote entry of projects adhering to quality control and standard operating procedure (QC/SOP) criteria, and automated output of alternative probe set algorithms for each project (see http://microarray.cnmcresearch.org/pgadatatable.asp).

The PEPR GeneChip data warehouse, and implementation of a dynamic time series query tool (SGQT) with graphical interface

PubMed Central

Chen, Josephine; Zhao, Po; Massaro, Donald; Clerch, Linda B.; Almon, Richard R.; DuBois, Debra C.; Jusko, William J.; Hoffman, Eric P.

2004-01-01

Publicly accessible DNA databases (genome browsers) are rapidly accelerating post-genomic research (see http://www.genome.ucsc.edu/), with integrated genomic DNA, gene structure, EST/ splicing and cross-species ortholog data. DNA databases have relatively low dimensionality; the genome is a linear code that anchors all associated data. In contrast, RNA expression and protein databases need to be able to handle very high dimensional data, with time, tissue, cell type and genes, as interrelated variables. The high dimensionality of microarray expression profile data, and the lack of a standard experimental platform have complicated the development of web-accessible databases and analytical tools. We have designed and implemented a public resource of expression profile data containing 1024 human, mouse and rat Affymetrix GeneChip expression profiles, generated in the same laboratory, and subject to the same quality and procedural controls (Public Expression Profiling Resource; PEPR). Our Oracle-based PEPR data warehouse includes a novel time series query analysis tool (SGQT), enabling dynamic generation of graphs and spreadsheets showing the action of any transcript of interest over time. In this report, we demonstrate the utility of this tool using a 27 time point, in vivo muscle regeneration series. This data warehouse and associated analysis tools provides access to multidimensional microarray data through web-based interfaces, both for download of all types of raw data for independent analysis, and also for straightforward gene-based queries. Planned implementations of PEPR will include web-based remote entry of projects adhering to quality control and standard operating procedure (QC/SOP) criteria, and automated output of alternative probe set algorithms for each project (see http://microarray.cnmcresearch.org/pgadatatable.asp). PMID:14681485

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...

Cross-Laboratory Analysis of Brain Cell Type Transcriptomes with Applications to Interpretation of Bulk Tissue Data

PubMed Central

Toker, Lilah; Rocco, Brad; Sibille, Etienne

2017-01-01

Establishing the molecular diversity of cell types is crucial for the study of the nervous system. We compiled a cross-laboratory database of mouse brain cell type-specific transcriptomes from 36 major cell types from across the mammalian brain using rigorously curated published data from pooled cell type microarray and single-cell RNA-sequencing (RNA-seq) studies. We used these data to identify cell type-specific marker genes, discovering a substantial number of novel markers, many of which we validated using computational and experimental approaches. We further demonstrate that summarized expression of marker gene sets (MGSs) in bulk tissue data can be used to estimate the relative cell type abundance across samples. To facilitate use of this expanding resource, we provide a user-friendly web interface at www.neuroexpresso.org. PMID:29204516

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.

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.

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.

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 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.

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.

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

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

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…

RNA-seq based transcriptomic map reveals new insights into mouse salivary gland development and maturation.

PubMed

Gluck, Christian; Min, Sangwon; Oyelakin, Akinsola; Smalley, Kirsten; Sinha, Satrajit; Romano, Rose-Anne

2016-11-16

Mouse models have served a valuable role in deciphering various facets of Salivary Gland (SG) biology, from normal developmental programs to diseased states. To facilitate such studies, gene expression profiling maps have been generated for various stages of SG organogenesis. However these prior studies fall short of capturing the transcriptional complexity due to the limited scope of gene-centric microarray-based technology. Compared to microarray, RNA-sequencing (RNA-seq) offers unbiased detection of novel transcripts, broader dynamic range and high specificity and sensitivity for detection of genes, transcripts, and differential gene expression. Although RNA-seq data, particularly under the auspices of the ENCODE project, have covered a large number of biological specimens, studies on the SG have been lacking. To better appreciate the wide spectrum of gene expression profiles, we isolated RNA from mouse submandibular salivary glands at different embryonic and adult stages. In parallel, we processed RNA-seq data for 24 organs and tissues obtained from the mouse ENCODE consortium and calculated the average gene expression values. To identify molecular players and pathways likely to be relevant for SG biology, we performed functional gene enrichment analysis, network construction and hierarchal clustering of the RNA-seq datasets obtained from different stages of SG development and maturation, and other mouse organs and tissues. Our bioinformatics-based data analysis not only reaffirmed known modulators of SG morphogenesis but revealed novel transcription factors and signaling pathways unique to mouse SG biology and function. Finally we demonstrated that the unique SG gene signature obtained from our mouse studies is also well conserved and can demarcate features of the human SG transcriptome that is different from other tissues. Our RNA-seq based Atlas has revealed a high-resolution cartographic view of the dynamic transcriptomic landscape of the mouse SG at various stages. These RNA-seq datasets will complement pre-existing microarray based datasets, including the Salivary Gland Molecular Anatomy Project by offering a broader systems-biology based perspective rather than the classical gene-centric view. Ultimately such resources will be valuable in providing a useful toolkit to better understand how the diverse cell population of the SG are organized and controlled during development and differentiation.

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.

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

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 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.

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

Comparison of Comparative Genomic Hybridization Technologies across Microarray Platforms

EPA Science Inventory

In the 2007 Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation (CNV) was analyzed ...

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray

PubMed Central

2010-01-01

Background Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Results Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. Conclusion All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues. PMID:20964859

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.

PubMed

Fenart, Stéphane; Ndong, Yves-Placide Assoumou; Duarte, Jorge; Rivière, Nathalie; Wilmer, Jeroen; van Wuytswinkel, Olivier; Lucau, Anca; Cariou, Emmanuelle; Neutelings, Godfrey; Gutierrez, Laurent; Chabbert, Brigitte; Guillot, Xavier; Tavernier, Reynald; Hawkins, Simon; Thomasset, Brigitte

2010-10-21

Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.

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.

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.

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.).

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.

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.

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.

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.

DGEM--a microarray gene expression database for primary human disease tissues.

PubMed

Xia, Yuni; Campen, Andrew; Rigsby, Dan; Guo, Ying; Feng, Xingdong; Su, Eric W; Palakal, Mathew; Li, Shuyu

2007-01-01

Gene expression patterns can reflect gene regulations in human tissues under normal or pathologic conditions. Gene expression profiling data from studies of primary human disease samples are particularly valuable since these studies often span many years in order to collect patient clinical information and achieve a large sample size. Disease-to-Gene Expression Mapper (DGEM) provides a beneficial community resource to access and analyze these data; it currently includes Affymetrix oligonucleotide array datasets for more than 40 human diseases and 1400 samples. The data are normalized to the same scale and stored in a relational database. A statistical-analysis pipeline was implemented to identify genes abnormally expressed in disease tissues or genes whose expressions are associated with clinical parameters such as cancer patient survival. Data-mining results can be queried through a web-based interface at http://dgem.dhcp.iupui.edu/. The query tool enables dynamic generation of graphs and tables that are further linked to major gene and pathway resources that connect the data to relevant biology, including Entrez Gene and Kyoto Encyclopedia of Genes and Genomes (KEGG). In summary, DGEM provides scientists and physicians a valuable tool to study disease mechanisms, to discover potential disease biomarkers for diagnosis and prognosis, and to identify novel gene targets for drug discovery. The source code is freely available for non-profit use, on request to the authors.

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.

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

CellFinder: a cell data repository

PubMed Central

Stachelscheid, Harald; Seltmann, Stefanie; Lekschas, Fritz; Fontaine, Jean-Fred; Mah, Nancy; Neves, Mariana; Andrade-Navarro, Miguel A.; Leser, Ulf; Kurtz, Andreas

2014-01-01

CellFinder (http://www.cellfinder.org) is a comprehensive one-stop resource for molecular data characterizing mammalian cells in different tissues and in different development stages. It is built from carefully selected data sets stemming from other curated databases and the biomedical literature. To date, CellFinder describes 3394 cell types and 50 951 cell lines. The database currently contains 3055 microscopic and anatomical images, 205 whole-genome expression profiles of 194 cell/tissue types from RNA-seq and microarrays and 553 905 protein expressions for 535 cells/tissues. Text mining of a corpus of >2000 publications followed by manual curation confirmed expression information on ∼900 proteins and genes. CellFinder’s data model is capable to seamlessly represent entities from single cells to the organ level, to incorporate mappings between homologous entities in different species and to describe processes of cell development and differentiation. Its ontological backbone currently consists of 204 741 ontology terms incorporated from 10 different ontologies unified under the novel CELDA ontology. CellFinder’s web portal allows searching, browsing and comparing the stored data, interactive construction of developmental trees and navigating the partonomic hierarchy of cells and tissues through a unique body browser designed for life scientists and clinicians. PMID:24304896

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.

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

Structural requirements of research tissue banks derived from standardized project surveillance.

PubMed

Herpel, E; Koleganova, N; Schreiber, B; Walter, B; Kalle, C V; Schirmacher, P

2012-07-01

Tissue banks constitute decisive and rate-limiting resource and technology platforms for basic and translational biomedical research, notably in the area of cancer. Thus, it is essential to plan and structure tissue banking and allocate resources according to research needs, but essential requirements are still incompletely defined. The tissue bank of the National Center of Tumor Diseases Heidelberg (NCT) was founded with the intention to provide tissues of optimal quality and to prioritize the realization of research projects. We analysed its structure and prospective project management registration as well as tracking records for all projects of the NCT tissue bank as of its start in 2005 in order to obtain information that may be relevant for tissue bank planning. All project proposals submitted to the NCT tissue bank (n = 681) were included in the study. For a detailed evaluation of provided services, only projects that were completed until July 2011 (n = 605) were analysed. For these 605 projects, NCT tissue bank provided 769 specific services. In all projects/services, we recorded project leader, type and amount of material provided, type of research (basic/translational), work load of project and project completion. Furthermore, all completed projects were tracked after 90 days according to a standard protocol to determine principal investigators' (PI) satisfaction and quality of the provided material. Until July 2011, 605 projects had been successfully completed as documented by material transfer agreement. Of the projects, 72.7 % addressed basic research, 22.3 % were translational research projects and 3 % concerned epidemiological research; 91 % (n = 546) concerned a single PI and the NTC tissue bank. For these projects, 769 specific services were provided. Of these services, 288 concerned providing formalin-fixed and paraffin-embedded (FFPE) tissue (extracts, full size sections), 126 providing fresh frozen materials (including fresh frozen sections), 137 providing tissue micro-array (TMA)-based sections and 199 providing immunohistochemical services. Project tracking demonstrated that all projects had started within 90 days after reception of the material by the PIs, and PI satisfaction with provided material exceeded 97 %. Standardized registration and tracking provides valuable structural information for planning and financing of tissue banks and allocation of resources. The high number of completed projects as well as high user satisfaction demonstrates that structuring of tissue banks should be preferably research-oriented and highly efficient. The comparable number of requests for FFPE and fresh frozen tissue as well as TMA-based services underpins the need for a broad approach in terms of methods and material types in order to fulfil research needs.

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

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.

Correlation of gene expression and contaminat concentrations in wild largescale suckers: a field-based study

USGS Publications Warehouse

Christiansen, Helena E.; Mehinto, Alvine C.; Yu, Fahong; Perry, Russell W.; Denslow, Nancy D.; Maule, Alec G.; Mesa, Matthew G.

2014-01-01

Toxic compounds such as organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether flame retardants (PBDEs) have been detected in fish, birds, and aquatic mammals that live in the Columbia River or use food resources from within the river. We developed a custom microarray for largescale suckers (Catostomus macrocheilus) and used it to investigate the molecular effects of contaminant exposure on wild fish in the Columbia River. Using Significance Analysis of Microarrays (SAM) we identified 72 probes representing 69 unique genes with expression patterns that correlated with hepatic tissue levels of OCs, PCBs, or PBDEs. These genes were involved in many biological processes previously shown to respond to contaminant exposure, including drug and lipid metabolism, apoptosis, cellular transport, oxidative stress, and cellular chaperone function. The relation between gene expression and contaminant concentration suggests that these genes may respond to environmental contaminant exposure and are promising candidates for further field and laboratory studies to develop biomarkers for monitoring exposure of wild fish to contaminant mixtures found in the Columbia River Basin. The array developed in this study could also be a useful tool for studies involving endangered sucker species and other sucker species used in contaminant research.

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.

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).

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.

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.

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

MIGS-GPU: Microarray Image Gridding and Segmentation on the GPU.

PubMed

Katsigiannis, Stamos; Zacharia, Eleni; Maroulis, Dimitris

2017-05-01

Complementary DNA (cDNA) microarray is a powerful tool for simultaneously studying the expression level of thousands of genes. Nevertheless, the analysis of microarray images remains an arduous and challenging task due to the poor quality of the images that often suffer from noise, artifacts, and uneven background. In this study, the MIGS-GPU [Microarray Image Gridding and Segmentation on Graphics Processing Unit (GPU)] software for gridding and segmenting microarray images is presented. MIGS-GPU's computations are performed on the GPU by means of the compute unified device architecture (CUDA) in order to achieve fast performance and increase the utilization of available system resources. Evaluation on both real and synthetic cDNA microarray images showed that MIGS-GPU provides better performance than state-of-the-art alternatives, while the proposed GPU implementation achieves significantly lower computational times compared to the respective CPU approaches. Consequently, MIGS-GPU can be an advantageous and useful tool for biomedical laboratories, offering a user-friendly interface that requires minimum input in order to run.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

PubMed Central

2012-01-01

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, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings. PMID:16964229

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.

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…

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.

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.

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

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.

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.

Salty dog, an SLC5 symporter, modulates Drosophila response to salt stress.

PubMed

Stergiopoulos, Konstantinos; Cabrero, Pablo; Davies, Shireen-Anne; Dow, Julian A T

2009-03-03

To regulate their internal environments, organisms must adapt to varying ion levels in their diet. Adult Drosophila were exposed to dietary salt stress, and their physiological, survival, and gene expression responses monitored. Insects continued to feed on NaCl-elevated diet, although levels >4% wt/vol ultimately proved fatal. Affymetrix microarray analysis of flies fed on diet containing elevated NaCl showed a phased response: the earliest response was widespread upregulation of immune genes, followed by upregulation of carbohydrate metabolism as the immune response was downregulated, then finally a switch to amino acid catabolism and inhibition of genes associated with the reproductive axis. Significantly, the online transcriptomic resource FlyAtlas reports that most of the modulated genes are predominantly expressed in hindgut or Malpighian (renal) tubule, implicating these excretory tissues as the major responders to salt stress. Three genes were selected for further study: the SLC5 symporter CG2196, the GLUT transporter CG6484, and the transcription factor sugarbabe (previously implicated in starvation and stress responses). Expression profiles predicted by microarray were validated by quantitative PCR (qPCR); expression was mapped to the alimentary canal by in situ hybridization. CG2196::eYFP overexpression constructs were localized to the basolateral membrane of the Malpighian (renal) tubules, and RNAi against CG2196 improved survival on high-salt diet, even when driven specifically to just principal cells of the Malpighian tubule, confirming both this tissue and this transporter as major determinants of survival upon salt stress. Accordingly, CG2196 was renamed salty dog (salt).

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.

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.

A novel strategy of integrated microarray analysis identifies CENPA, CDK1 and CDC20 as a cluster of diagnostic biomarkers in lung adenocarcinoma.

PubMed

Liu, Wan-Ting; Wang, Yang; Zhang, Jing; Ye, Fei; Huang, Xiao-Hui; Li, Bin; He, Qing-Yu

2018-07-01

Lung adenocarcinoma (LAC) is the most lethal cancer and the leading cause of cancer-related death worldwide. The identification of meaningful clusters of co-expressed genes or representative biomarkers may help improve the accuracy of LAC diagnoses. Public databases, such as the Gene Expression Omnibus (GEO), provide rich resources of valuable information for clinics, however, the integration of multiple microarray datasets from various platforms and institutes remained a challenge. To determine potential indicators of LAC, we performed genome-wide relative significance (GWRS), genome-wide global significance (GWGS) and support vector machine (SVM) analyses progressively to identify robust gene biomarker signatures from 5 different microarray datasets that included 330 samples. The top 200 genes with robust signatures were selected for integrative analysis according to "guilt-by-association" methods, including protein-protein interaction (PPI) analysis and gene co-expression analysis. Of these 200 genes, only 10 genes showed both intensive PPI network and high gene co-expression correlation (r > 0.8). IPA analysis of this regulatory networks suggested that the cell cycle process is a crucial determinant of LAC. CENPA, as well as two linked hub genes CDK1 and CDC20, are determined to be potential indicators of LAC. Immunohistochemical staining showed that CENPA, CDK1 and CDC20 were highly expressed in LAC cancer tissue with co-expression patterns. A Cox regression model indicated that LAC patients with CENPA + /CDK1 + and CENPA + /CDC20 + were high-risk groups in terms of overall survival. In conclusion, our integrated microarray analysis demonstrated that CENPA, CDK1 and CDC20 might serve as novel cluster of prognostic biomarkers for LAC, and the cooperative unit of three genes provides a technically simple approach for identification of LAC patients. Copyright © 2018 Elsevier B.V. All rights reserved.

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 ).

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

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens

PubMed Central

Pardo, Belén G; Fernández, Carlos; Millán, Adrián; Bouza, Carmen; Vázquez-López, Araceli; Vera, Manuel; Alvarez-Dios, José A; Calaza, Manuel; Gómez-Tato, Antonio; Vázquez, María; Cabaleiro, Santiago; Magariños, Beatriz; Lemos, Manuel L; Leiro, José M; Martínez, Paulino

2008-01-01

Background The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. Results A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. Conclusion A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot. PMID:18817567

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.

A study of metaheuristic algorithms for high dimensional feature selection on microarray data

NASA Astrophysics Data System (ADS)

Dankolo, Muhammad Nasiru; Radzi, Nor Haizan Mohamed; Sallehuddin, Roselina; Mustaffa, Noorfa Haszlinna

2017-11-01

Microarray systems enable experts to examine gene profile at molecular level using machine learning algorithms. It increases the potentials of classification and diagnosis of many diseases at gene expression level. Though, numerous difficulties may affect the efficiency of machine learning algorithms which includes vast number of genes features comprised in the original data. Many of these features may be unrelated to the intended analysis. Therefore, feature selection is necessary to be performed in the data pre-processing. Many feature selection algorithms are developed and applied on microarray which including the metaheuristic optimization algorithms. This paper discusses the application of the metaheuristics algorithms for feature selection in microarray dataset. This study reveals that, the algorithms have yield an interesting result with limited resources thereby saving computational expenses of machine learning algorithms.

Comparative biomarker expression and RNA integrity in biospecimens derived from radical retropubic and robot-assisted laparoscopic prostatectomies.

PubMed

Ricciardelli, Carmela; Bianco-Miotto, Tina; Jindal, Shalini; Dodd, Thomas J; Cohen, Penelope A; Marshall, Villis R; Sutherland, Peter D; Samaratunga, Hemamali; Kench, James G; Dong, Ying; Wang, Hong; Clements, Judith A; Risbridger, Gail P; Sutherland, Robert L; Tilley, Wayne D; Horsfall, David J

2010-07-01

Knowledge of preanalytic conditions that biospecimens are subjected to is critically important because novel surgical procedures, tissue sampling, handling, and storage might affect biomarker expression or invalidate tissue samples as analytes for some technologies. We investigated differences in RNA quality, gene expression by quantitative real-time PCR, and immunoreactive protein expression of selected prostate cancer biomarkers between tissues from retropubic radical prostatectomy (RRP) and robot-assisted laparoscopic prostatectomy (RALP). Sections of tissue microarray of 23 RALP and 22 RRP samples were stained with antibodies to androgen receptor (AR) and prostate-specific antigen (PSA) as intersite controls, and 14 other candidate biomarkers of research interest to three laboratories within the Australian Prostate Cancer BioResource tissue banking network. Quantitative real-time PCR was done for AR, PSA (KLK3), KLK2, KLK4, and HIF1A on RNA extracted from five RALP and five RRP frozen tissue cores. No histologic differences were observed between RALP and RRP tissue. Biomarker staining grouped these samples into those with increased (PSA, CK8/18, CKHMW, KLK4), decreased (KLK2, KLK14), or no change in expression (AR, ghrelin, Ki67, PCNA, VEGF-C, PAR2, YB1, p63, versican, and chondroitin 0-sulfate) in RALP compared with RRP tissue. No difference in RNA quality or gene expression was detected between RALP and RRP tissue. Changes in biomarker expression between RALP and RRP tissue exist at the immunoreactive protein level, but the etiology is unclear. Future studies should account for changes in biomarker expression when using RALP tissues, and mixed cohorts of RALP and RRP tissue should be avoided.

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 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

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.

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.

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

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

Workflows for microarray data processing in the Kepler environment.

PubMed

Stropp, Thomas; McPhillips, Timothy; Ludäscher, Bertram; Bieda, Mark

2012-05-17

Microarray data analysis has been the subject of extensive and ongoing pipeline development due to its complexity, the availability of several options at each analysis step, and the development of new analysis demands, including integration with new data sources. Bioinformatics pipelines are usually custom built for different applications, making them typically difficult to modify, extend and repurpose. Scientific workflow systems are intended to address these issues by providing general-purpose frameworks in which to develop and execute such pipelines. The Kepler workflow environment is a well-established system under continual development that is employed in several areas of scientific research. Kepler provides a flexible graphical interface, featuring clear display of parameter values, for design and modification of workflows. It has capabilities for developing novel computational components in the R, Python, and Java programming languages, all of which are widely used for bioinformatics algorithm development, along with capabilities for invoking external applications and using web services. We developed a series of fully functional bioinformatics pipelines addressing common tasks in microarray processing in the Kepler workflow environment. These pipelines consist of a set of tools for GFF file processing of NimbleGen chromatin immunoprecipitation on microarray (ChIP-chip) datasets and more comprehensive workflows for Affymetrix gene expression microarray bioinformatics and basic primer design for PCR experiments, which are often used to validate microarray results. Although functional in themselves, these workflows can be easily customized, extended, or repurposed to match the needs of specific projects and are designed to be a toolkit and starting point for specific applications. These workflows illustrate a workflow programming paradigm focusing on local resources (programs and data) and therefore are close to traditional shell scripting or R/BioConductor scripting approaches to pipeline design. Finally, we suggest that microarray data processing task workflows may provide a basis for future example-based comparison of different workflow systems. We provide a set of tools and complete workflows for microarray data analysis in the Kepler environment, which has the advantages of offering graphical, clear display of conceptual steps and parameters and the ability to easily integrate other resources such as remote data and web services.

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

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

Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum.

PubMed

Page, Robert B; Monaghan, James R; Samuels, Amy K; Smith, Jeramiah J; Beachy, Christopher K; Voss, S Randal

2007-02-01

Ambystomatid salamanders offer several advantages for endocrine disruption research, including genomic and bioinformatics resources, an accessible laboratory model (Ambystoma mexicanum), and natural lineages that are broadly distributed among North American habitats. We used microarray analysis to measure the relative abundance of transcripts isolated from A. mexicanum epidermis (skin) after exogenous application of thyroid hormone (TH). Only one gene had a >2-fold change in transcript abundance after 2 days of TH treatment. However, hundreds of genes showed significantly different transcript levels at days 12 and 28 in comparison to day 0. A list of 123 TH-responsive genes was identified using statistical, BLAST, and fold level criteria. Cluster analysis identified two groups of genes with similar transcription patterns: up-regulated versus down-regulated. Most notably, several keratins exhibited dramatic (1000 fold) increases or decreases in transcript abundance. Keratin gene expression changes coincided with morphological remodeling of epithelial tissues. This suggests that keratin loci can be developed as sensitive biomarkers to assay temporal disruptions of larval-to-adult gene expression programs. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander, thus setting the stage for future investigations of TH disruption in the Mexican axolotl and other salamanders of the genus Ambystoma.

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.

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.

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

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

MicroArray Facility: a laboratory information management system with extended support for Nylon based technologies.

PubMed

Honoré, Paul; Granjeaud, Samuel; Tagett, Rebecca; Deraco, Stéphane; Beaudoing, Emmanuel; Rougemont, Jacques; Debono, Stéphane; Hingamp, Pascal

2006-09-20

High throughput gene expression profiling (GEP) is becoming a routine technique in life science laboratories. With experimental designs that repeatedly span thousands of genes and hundreds of samples, relying on a dedicated database infrastructure is no longer an option.GEP technology is a fast moving target, with new approaches constantly broadening the field diversity. This technology heterogeneity, compounded by the informatics complexity of GEP databases, means that software developments have so far focused on mainstream techniques, leaving less typical yet established techniques such as Nylon microarrays at best partially supported. MAF (MicroArray Facility) is the laboratory database system we have developed for managing the design, production and hybridization of spotted microarrays. Although it can support the widely used glass microarrays and oligo-chips, MAF was designed with the specific idiosyncrasies of Nylon based microarrays in mind. Notably single channel radioactive probes, microarray stripping and reuse, vector control hybridizations and spike-in controls are all natively supported by the software suite. MicroArray Facility is MIAME supportive and dynamically provides feedback on missing annotations to help users estimate effective MIAME compliance. Genomic data such as clone identifiers and gene symbols are also directly annotated by MAF software using standard public resources. The MAGE-ML data format is implemented for full data export. Journalized database operations (audit tracking), data anonymization, material traceability and user/project level confidentiality policies are also managed by MAF. MicroArray Facility is a complete data management system for microarray producers and end-users. Particular care has been devoted to adequately model Nylon based microarrays. The MAF system, developed and implemented in both private and academic environments, has proved a robust solution for shared facilities and industry service providers alike.

MicroArray Facility: a laboratory information management system with extended support for Nylon based technologies

PubMed Central

Honoré, Paul; Granjeaud, Samuel; Tagett, Rebecca; Deraco, Stéphane; Beaudoing, Emmanuel; Rougemont, Jacques; Debono, Stéphane; Hingamp, Pascal

2006-01-01

Background High throughput gene expression profiling (GEP) is becoming a routine technique in life science laboratories. With experimental designs that repeatedly span thousands of genes and hundreds of samples, relying on a dedicated database infrastructure is no longer an option. GEP technology is a fast moving target, with new approaches constantly broadening the field diversity. This technology heterogeneity, compounded by the informatics complexity of GEP databases, means that software developments have so far focused on mainstream techniques, leaving less typical yet established techniques such as Nylon microarrays at best partially supported. Results MAF (MicroArray Facility) is the laboratory database system we have developed for managing the design, production and hybridization of spotted microarrays. Although it can support the widely used glass microarrays and oligo-chips, MAF was designed with the specific idiosyncrasies of Nylon based microarrays in mind. Notably single channel radioactive probes, microarray stripping and reuse, vector control hybridizations and spike-in controls are all natively supported by the software suite. MicroArray Facility is MIAME supportive and dynamically provides feedback on missing annotations to help users estimate effective MIAME compliance. Genomic data such as clone identifiers and gene symbols are also directly annotated by MAF software using standard public resources. The MAGE-ML data format is implemented for full data export. Journalized database operations (audit tracking), data anonymization, material traceability and user/project level confidentiality policies are also managed by MAF. Conclusion MicroArray Facility is a complete data management system for microarray producers and end-users. Particular care has been devoted to adequately model Nylon based microarrays. The MAF system, developed and implemented in both private and academic environments, has proved a robust solution for shared facilities and industry service providers alike. PMID:16987406

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

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.

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...

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

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.

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

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.

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.

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.

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

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.

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.

Improved microarray methods for profiling the yeast knockout strain collection

PubMed Central

Yuan, Daniel S.; Pan, Xuewen; Ooi, Siew Loon; Peyser, Brian D.; Spencer, Forrest A.; Irizarry, Rafael A.; Boeke, Jef D.

2005-01-01

A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens. PMID:15994458

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.

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

PubMed Central

2013-01-01

Background The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. Conclusions This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported. PMID:24168212

mRNA-Seq and microarray development for the Grooved Carpet shell clam, Ruditapes decussatus: a functional approach to unravel host-parasite interaction.

PubMed

Leite, Ricardo B; Milan, Massimo; Coppe, Alessandro; Bortoluzzi, Stefania; dos Anjos, António; Reinhardt, Richard; Saavedra, Carlos; Patarnello, Tomaso; Cancela, M Leonor; Bargelloni, Luca

2013-10-29

The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

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.

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.

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.

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

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

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

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.

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

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

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

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

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

National Mesothelioma Virtual Bank: a standard based biospecimen and clinical data resource to enhance translational research.

PubMed

Amin, Waqas; Parwani, Anil V; Schmandt, Linda; Mohanty, Sambit K; Farhat, Ghada; Pople, Andrew K; Winters, Sharon B; Whelan, Nancy B; Schneider, Althea M; Milnes, John T; Valdivieso, Federico A; Feldman, Michael; Pass, Harvey I; Dhir, Rajiv; Melamed, Jonathan; Becich, Michael J

2008-08-13

Advances in translational research have led to the need for well characterized biospecimens for research. The National Mesothelioma Virtual Bank is an initiative which collects annotated datasets relevant to human mesothelioma to develop an enterprising biospecimen resource to fulfill researchers' need. The National Mesothelioma Virtual Bank architecture is based on three major components: (a) common data elements (based on College of American Pathologists protocol and National North American Association of Central Cancer Registries standards), (b) clinical and epidemiologic data annotation, and (c) data query tools. These tools work interoperably to standardize the entire process of annotation. The National Mesothelioma Virtual Bank tool is based upon the caTISSUE Clinical Annotation Engine, developed by the University of Pittsburgh in cooperation with the Cancer Biomedical Informatics Grid (caBIG, see http://cabig.nci.nih.gov). This application provides a web-based system for annotating, importing and searching mesothelioma cases. The underlying information model is constructed utilizing Unified Modeling Language class diagrams, hierarchical relationships and Enterprise Architect software. The database provides researchers real-time access to richly annotated specimens and integral information related to mesothelioma. The data disclosed is tightly regulated depending upon users' authorization and depending on the participating institute that is amenable to the local Institutional Review Board and regulation committee reviews. The National Mesothelioma Virtual Bank currently has over 600 annotated cases available for researchers that include paraffin embedded tissues, tissue microarrays, serum and genomic DNA. The National Mesothelioma Virtual Bank is a virtual biospecimen registry with robust translational biomedical informatics support to facilitate basic science, clinical, and translational research. Furthermore, it protects patient privacy by disclosing only de-identified datasets to assure that biospecimens can be made accessible to researchers.

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

Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern

PubMed Central

Matigian, Nicholas; Brooke, Gary; Zaibak, Faten; Rossetti, Tony; Kollar, Katarina; Pelekanos, Rebecca; Heazlewood, Celena; Mackay-Sim, Alan; Wells, Christine A.; Atkinson, Kerry

2014-01-01

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues. PMID:26484151

Metadata management and semantics in microarray repositories.

PubMed

Kocabaş, F; Can, T; Baykal, N

2011-12-01

The number of microarray and other high-throughput experiments on primary repositories keeps increasing as do the size and complexity of the results in response to biomedical investigations. Initiatives have been started on standardization of content, object model, exchange format and ontology. However, there are backlogs and inability to exchange data between microarray repositories, which indicate that there is a great need for a standard format and data management. We have introduced a metadata framework that includes a metadata card and semantic nets that make experimental results visible, understandable and usable. These are encoded in syntax encoding schemes and represented in RDF (Resource Description Frame-word), can be integrated with other metadata cards and semantic nets, and can be exchanged, shared and queried. We demonstrated the performance and potential benefits through a case study on a selected microarray repository. We concluded that the backlogs can be reduced and that exchange of information and asking of knowledge discovery questions can become possible with the use of this metadata framework.

DNA Microarray Wet Lab Simulation Brings Genomics into the High School Curriculum

PubMed Central

Zanta, Carolyn A.; Heyer, Laurie J.; Kittinger, Ben; Gabric, Kathleen M.; Adler, Leslie

2006-01-01

We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH indicators, which offer many ideal teaching characteristics. The simulation requires no specialized equipment, is very inexpensive, is very reliable, and takes very little preparation time. Student and teacher assessment data indicate the simulation is popular with both groups, and students show significant learning gains. We include many resources with this publication, including all prelab introductory materials (e.g., a paper microarray activity), the student handouts, teachers notes, and pre- and postassessment tools. We did not test the simulation on other student populations, but based on teacher feedback, the simulation also may fit well in community college and in introductory and nonmajors' college biology curricula. PMID:17146040

DNA microarray wet lab simulation brings genomics into the high school curriculum.

PubMed

Campbell, A Malcolm; Zanta, Carolyn A; Heyer, Laurie J; Kittinger, Ben; Gabric, Kathleen M; Adler, Leslie; Schulz, Barbara

2006-01-01

We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH indicators, which offer many ideal teaching characteristics. The simulation requires no specialized equipment, is very inexpensive, is very reliable, and takes very little preparation time. Student and teacher assessment data indicate the simulation is popular with both groups, and students show significant learning gains. We include many resources with this publication, including all prelab introductory materials (e.g., a paper microarray activity), the student handouts, teachers notes, and pre- and postassessment tools. We did not test the simulation on other student populations, but based on teacher feedback, the simulation also may fit well in community college and in introductory and nonmajors' college biology curricula.

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.

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

[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.

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

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

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

The MGED Ontology: a resource for semantics-based description of microarray experiments.

PubMed

Whetzel, Patricia L; Parkinson, Helen; Causton, Helen C; Fan, Liju; Fostel, Jennifer; Fragoso, Gilberto; Game, Laurence; Heiskanen, Mervi; Morrison, Norman; Rocca-Serra, Philippe; Sansone, Susanna-Assunta; Taylor, Chris; White, Joseph; Stoeckert, Christian J

2006-04-01

The generation of large amounts of microarray data and the need to share these data bring challenges for both data management and annotation and highlights the need for standards. MIAME specifies the minimum information needed to describe a microarray experiment and the Microarray Gene Expression Object Model (MAGE-OM) and resulting MAGE-ML provide a mechanism to standardize data representation for data exchange, however a common terminology for data annotation is needed to support these standards. Here we describe the MGED Ontology (MO) developed by the Ontology Working Group of the Microarray Gene Expression Data (MGED) Society. The MO provides terms for annotating all aspects of a microarray experiment from the design of the experiment and array layout, through to the preparation of the biological sample and the protocols used to hybridize the RNA and analyze the data. The MO was developed to provide terms for annotating experiments in line with the MIAME guidelines, i.e. to provide the semantics to describe a microarray experiment according to the concepts specified in MIAME. The MO does not attempt to incorporate terms from existing ontologies, e.g. those that deal with anatomical parts or developmental stages terms, but provides a framework to reference terms in other ontologies and therefore facilitates the use of ontologies in microarray data annotation. The MGED Ontology version.1.2.0 is available as a file in both DAML and OWL formats at http://mged.sourceforge.net/ontologies/index.php. Release notes and annotation examples are provided. The MO is also provided via the NCICB's Enterprise Vocabulary System (http://nciterms.nci.nih.gov/NCIBrowser/Dictionary.do). Stoeckrt@pcbi.upenn.edu Supplementary data are available at Bioinformatics online.

Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species.

PubMed

Ahn, Soohyoun; Kulis, David M; Erdner, Deana L; Anderson, Donald M; Walt, David R

2006-09-01

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 mum) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.

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

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.

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.

Efficient mutation identification in zebrafish by microarray capturing and next generation sequencing.

PubMed

Bontems, Franck; Baerlocher, Loic; Mehenni, Sabrina; Bahechar, Ilham; Farinelli, Laurent; Dosch, Roland

2011-02-18

Fish models like medaka, stickleback or zebrafish provide a valuable resource to study vertebrate genes. However, finding genetic variants e.g. mutations in the genome is still arduous. Here we used a combination of microarray capturing and next generation sequencing to identify the affected gene in the mozartkugelp11cv (mzlp11cv) mutant zebrafish. We discovered a 31-bp deletion in macf1 demonstrating the potential of this technique to efficiently isolate mutations in a vertebrate genome. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

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.

Genomic resources for songbird research and their use in characterizing gene expression during brain development

PubMed Central

Li, XiaoChing; Wang, Xiu-Jie; Tannenhauser, Jonathan; Podell, Sheila; Mukherjee, Piali; Hertel, Moritz; Biane, Jeremy; Masuda, Shoko; Nottebohm, Fernando; Gaasterland, Terry

2007-01-01

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3′ ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30–50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis. PMID:17426146

AbMiner: a bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies.

PubMed

Major, Sylvia M; Nishizuka, Satoshi; Morita, Daisaku; Rowland, Rick; Sunshine, Margot; Shankavaram, Uma; Washburn, Frank; Asin, Daniel; Kouros-Mehr, Hosein; Kane, David; Weinstein, John N

2006-04-06

Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60) and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different tissues of origin. AbMiner is a relational database that provides extensive information from our own laboratory and other sources on more than 600 available antibodies and the genes that encode the antibodies' antigens. The data will be made freely available at http://discover.nci.nih.gov/abminer.

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

Tissues from the irradiated dog/mouse archive

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

Gayle Woloschak

The purpose of this project is to organize the databases/information and organize and move the tissues from the long-term dog (4,000 dogs) and mouse (over 30,000 mice) radiation experiments done at Argonne National Laboratory during the 1970's and 80's to Northwestern University. These studies were done with the intention of understanding the effects of exposure to radiation at a variety of different doses, dose-rates, and radiation qualities on end-points such as life-shortening, carcinogenesis, cause of death, shifts in disease incidence and other biological parameters. Organ and tissue samples from these animals including cancers, metastases and other significant degenerative and inflammatorymore » lesions and those in a regular protocol of normal tissues were preserved in paraffin blocks, tissue impressions and sections and represent a great resource for the radiation biology community. These collections are particularly significant since these experiments are not likely to be repeated because of the extreme cost of monies and time for such large-scale animal studies. The long-term goal is to make these tissues and databases available to the wider scientific community so that questions such as tissue sensitivity, early and late effects, low dose and protracted dose responses of normal and tumor tissues, etc. can be examined and defined. Recent advances in biology particularly at the subcellular and molecular level now permit microarray-based gene expression array analyses from paraffin-embedded tissues (where RNA samples are significantly degraded), synchrotron-based studies of metal and other elemental distribution patterns in tissues, PCR-based analyses for mutation detection, and other similar approaches that were not available when the long¬ term animal studies were designed and initiated. Understanding the basis and progression of radiation damage should also permit rational approaches to prevention and mitigation of those damages. Therefore, as stated earlier, these tissues and their related documentation, represent a significant resource for future studies. For this project, we propose to accomplish the following objectives: (1) inventory and organize the tissues, blood smears, wet-tissues and paper-¬based information that is available in the tissue bank at Argonne National Laboratory; (2) convert the existing Oracle database of the mouse studies to MS Access( the dog data is already in this format which is far more user friendly and widely used in business and research) , (3) move the remaining samples and documentation from dogs that had been transferred from ANL to New Mexico (in Dr. F. Hahn's care) to Northwestern University and add these to the inventory; (4) move the tissues and Access database at Argonne National Laboratory to Northwestern University.« less

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

[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.

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.

SATRAT: Staphylococcus aureus transcript regulatory network analysis tool.

PubMed

Gopal, Tamilselvi; Nagarajan, Vijayaraj; Elasri, Mohamed O

2015-01-01

Staphylococcus aureus is a commensal organism that primarily colonizes the nose of healthy individuals. S. aureus causes a spectrum of infections that range from skin and soft-tissue infections to fatal invasive diseases. S. aureus uses a large number of virulence factors that are regulated in a coordinated fashion. The complex regulatory mechanisms have been investigated in numerous high-throughput experiments. Access to this data is critical to studying this pathogen. Previously, we developed a compilation of microarray experimental data to enable researchers to search, browse, compare, and contrast transcript profiles. We have substantially updated this database and have built a novel exploratory tool-SATRAT-the S. aureus transcript regulatory network analysis tool, based on the updated database. This tool is capable of performing deep searches using a query and generating an interactive regulatory network based on associations among the regulators of any query gene. We believe this integrated regulatory network analysis tool would help researchers explore the missing links and identify novel pathways that regulate virulence in S. aureus. Also, the data model and the network generation code used to build this resource is open sourced, enabling researchers to build similar resources for other bacterial systems.

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

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.

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

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.

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.

Adaptations to Endosymbiosis in a Cnidarian-Dinoflagellate Association: Differential Gene Expression and Specific Gene Duplications

PubMed Central

Magnone, Virginie; Allemand, Denis; Furla, Paola; Sabourault, Cécile

2011-01-01

Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion), which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays) from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones) or aposymbiotic (also called bleached) A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm). A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i) a key vitamin K–dependant process involved in the dinoflagellate-cnidarian recognition; ii) two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii) host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both in the symbiotic state and in the gastroderm. Our results thus offer new insight into the inter-partner signaling required for the physiological mechanisms of the symbiosis that is crucial for coral health. PMID:21811417

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.

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

Implementation of GenePattern within the Stanford Microarray Database.

PubMed

Hubble, Jeremy; Demeter, Janos; Jin, Heng; Mao, Maria; Nitzberg, Michael; Reddy, T B K; Wymore, Farrell; Zachariah, Zachariah K; Sherlock, Gavin; Ball, Catherine A

2009-01-01

Hundreds of researchers across the world use the Stanford Microarray Database (SMD; http://smd.stanford.edu/) to store, annotate, view, analyze and share microarray data. In addition to providing registered users at Stanford access to their own data, SMD also provides access to public data, and tools with which to analyze those data, to any public user anywhere in the world. Previously, the addition of new microarray data analysis tools to SMD has been limited by available engineering resources, and in addition, the existing suite of tools did not provide a simple way to design, execute and share analysis pipelines, or to document such pipelines for the purposes of publication. To address this, we have incorporated the GenePattern software package directly into SMD, providing access to many new analysis tools, as well as a plug-in architecture that allows users to directly integrate and share additional tools through SMD. In this article, we describe our implementation of the GenePattern microarray analysis software package into the SMD code base. This extension is available with the SMD source code that is fully and freely available to others under an Open Source license, enabling other groups to create a local installation of SMD with an enriched data analysis capability.

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

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Allen Brain Atlas: an integrated spatio-temporal portal for exploring the central nervous system

PubMed Central

Sunkin, Susan M.; Ng, Lydia; Lau, Chris; Dolbeare, Tim; Gilbert, Terri L.; Thompson, Carol L.; Hawrylycz, Michael; Dang, Chinh

2013-01-01

The Allen Brain Atlas (http://www.brain-map.org) provides a unique online public resource integrating extensive gene expression data, connectivity data and neuroanatomical information with powerful search and viewing tools for the adult and developing brain in mouse, human and non-human primate. Here, we review the resources available at the Allen Brain Atlas, describing each product and data type [such as in situ hybridization (ISH) and supporting histology, microarray, RNA sequencing, reference atlases, projection mapping and magnetic resonance imaging]. In addition, standardized and unique features in the web applications are described that enable users to search and mine the various data sets. Features include both simple and sophisticated methods for gene searches, colorimetric and fluorescent ISH image viewers, graphical displays of ISH, microarray and RNA sequencing data, Brain Explorer software for 3D navigation of anatomy and gene expression, and an interactive reference atlas viewer. In addition, cross data set searches enable users to query multiple Allen Brain Atlas data sets simultaneously. All of the Allen Brain Atlas resources can be accessed through the Allen Brain Atlas data portal. PMID:23193282

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

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.

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.

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.

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

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.

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.

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

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

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

Genomic resources in fruit plants: an assessment of current status.

PubMed

Rai, Manoj K; Shekhawat, N S

2015-01-01

The availability of many genomic resources such as genome sequences, functional genomics resources including microarrays and RNA-seq, sufficient numbers of molecular markers, express sequence tags (ESTs) and high-density genetic maps is causing a rapid acceleration of genetics and genomic research of many fruit plants. This is leading to an increase in our knowledge of the genes that are linked to many horticultural and agronomically important traits. Recently, some progress has also been made on the identification and functional analysis of miRNAs in some fruit plants. This is one of the most active research fields in plant sciences. The last decade has witnessed development of genomic resources in many fruit plants such as apple, banana, citrus, grapes, papaya, pears, strawberry etc.; however, many of them are still not being exploited. Furthermore, owing to lack of resources, infrastructure and research facilities in many lesser-developed countries, development of genomic resources in many underutilized or less-studied fruit crops, which grow in these countries, is limited. Thus, research emphasis should be given to those fruit crops for which genomic resources are relatively scarce. The development of genomic databases of these less-studied fruit crops will enable biotechnologists to identify target genes that underlie key horticultural and agronomical traits. This review presents an overview of the current status of the development of genomic resources in fruit plants with the main emphasis being on genome sequencing, EST resources, functional genomics resources including microarray and RNA-seq, identification of quantitative trait loci and construction of genetic maps as well as efforts made on the identification and functional analysis of miRNAs in fruit plants.

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

Psychiatric Brain Banking: Three Perspectives on Current Trends and Future Directions

PubMed Central

Deep-Soboslay, Amy; Benes, Francine M.; Haroutunian, Vahram; Ellis, Justin K.; Kleinman, Joel E.; Hyde, Thomas M.

2011-01-01

Introduction The study of postmortem human brain tissue is central to the advancement of the neurobiological studies of psychiatric illness, particularly for the study of brain-specific isoforms and molecules. Methods The state-of-the-art methods and recommendations for maintaining a successful brain bank for psychiatric disorders are discussed, using the convergence of viewpoints from three brain collections, the National Institute of Mental Health Brain Collection (NIMH), the Harvard Brain Tissue Resource Center (HBTRC), and the Mt. Sinai School of Medicine Brain Bank (MSSM-BB), with diverse research interests and divergent approaches to tissue acquisition. Results While the NIMH obtains donations from medical examiners for its collection, and places particular emphasis on clinical diagnosis, toxicology, and building lifespan control cohorts, the HBTRC is uniquely designed as a repository whose sole purpose is to collect large-volume, high quality brain tissue from community-based donors based on relationships across an expansive nationwide network, and places emphasis on the accessibility of its bank in disseminating tissue and related data to research groups worldwide. The MSSM-BB collection has shown that, with dedication, prospective recruitment is a successful approach to tissue donation, and places particular emphasis on rigorous clinical diagnosis through antemortem contact with donors. The MSSM-BB places great importance on stereological tissue sampling methods for neuroanatomical studies, and frozen tissue sampling approaches that enable multiple assessments (RNA, DNA, protein, enzyme activity, binding, etc.) of the same tissue block. Promising scientific approaches for elucidating the molecular and cellular pathways in brain that may contribute to schizophrenia and/or bipolar disorder, such as cell culture techniques and microarray-based gene expression and genotyping studies are briefly discussed. Conclusions Despite unique perspectives from three established brain collections, there is a consensus that (1) diverse strategies for tissue acquisition, (2) rigor in tissue and diagnostic characterization, (3) the importance of sample accessibility, and (4) continual application of innovative scientific approaches to the study of brain tissue are all integral to the success and future of psychiatric brain banking. The future of neuropsychiatric research depends upon in the availability of high quality brain specimens from large numbers of subjects, including non-psychiatric controls. PMID:20673875

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

Bioinformatics and Microarray Data Analysis on the Cloud.

PubMed

Calabrese, Barbara; Cannataro, Mario

2016-01-01

High-throughput platforms such as microarray, mass spectrometry, and next-generation sequencing are producing an increasing volume of omics data that needs large data storage and computing power. Cloud computing offers massive scalable computing and storage, data sharing, on-demand anytime and anywhere access to resources and applications, and thus, it may represent the key technology for facing those issues. In fact, in the recent years it has been adopted for the deployment of different bioinformatics solutions and services both in academia and in the industry. Although this, cloud computing presents several issues regarding the security and privacy of data, that are particularly important when analyzing patients data, such as in personalized medicine. This chapter reviews main academic and industrial cloud-based bioinformatics solutions; with a special focus on microarray data analysis solutions and underlines main issues and problems related to the use of such platforms for the storage and analysis of patients data.

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.

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.

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.

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

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

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.

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

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

Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks.

PubMed

Stutzer, Christian; van Zyl, Willem A; Olivier, Nicholas A; Richards, Sabine; Maritz-Olivier, Christine

2013-06-01

The southern cattle tick, Rhipicephalus microplus, is an economically important pest, especially for resource-poor countries, both as a highly adaptive invasive species and prominent vector of disease. The increasing prevalence of resistance to chemical acaricides and variable efficacy of current tick vaccine candidates highlight the need for more effective control methods. In the absence of a fully annotated genome, the wealth of available expressed sequence tag sequence data for this species presents a unique opportunity to study the genes that are expressed in tissues involved in blood meal acquisition, digestion and reproduction during feeding. Utilising a custom oligonucleotide microarray designed from available singletons (BmiGI Version 2.1) and expressed sequence tag sequences of R. microplus, the expression profiles in feeding adult female midgut, salivary glands and ovarian tissues were compared. From 13,456 assembled transcripts, 588 genes expressed in all three tissues were identified from fed adult females 20 days post infestation. The greatest complement of genes relate to translation and protein turnover. Additionally, a number of unique transcripts were identified for each tissue that relate well to their respective physiological/biological function/role(s). These transcripts include secreted anti-hemostatics and defense proteins from the salivary glands for acquisition of a blood meal, proteases as well as enzymes and transporters for digestion and nutrient acquisition from ingested blood in the midgut, and finally proteins and associated factors involved in DNA replication and cell-cycle control for oogenesis in the ovaries. Comparative analyses of adult female tissues during feeding enabled the identification of a catalogue of transcripts that may be essential for successful feeding and reproduction in the cattle tick, R. microplus. Future studies will increase our understanding of basic tick biology, allowing the identification of shared proteins/pathways among different tissues that may offer novel targets for the development of new tick control strategies. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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...

Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: a genomic resource for studying agricultural pests.

PubMed

Noda, Hiroaki; Kawai, Sawako; Koizumi, Yoko; Matsui, Kageaki; Zhang, Qiang; Furukawa, Shigetoyo; Shimomura, Michihiko; Mita, Kazuei

2008-03-03

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is a serious insect pests of rice plants. Major means of BPH control are application of agricultural chemicals and cultivation of BPH resistant rice varieties. Nevertheless, BPH strains that are resistant to agricultural chemicals have developed, and BPH strains have appeared that are virulent against the resistant rice varieties. Expressed sequence tag (EST) analysis and related applications are useful to elucidate the mechanisms of resistance and virulence and to reveal physiological aspects of this non-model insect, with its poorly understood genetic background. More than 37,000 high-quality ESTs, excluding sequences of mitochondrial genome, microbial genomes, and rDNA, have been produced from 18 libraries of various BPH tissues and stages. About 10,200 clusters have been made from whole EST sequences, with average EST size of 627 bp. Among the top ten most abundantly expressed genes, three are unique and show no homology in BLAST searches. The actin gene was highly expressed in BPH, especially in the thorax. Tissue-specifically expressed genes were extracted based on the expression frequency among the libraries. An EST database is available at our web site. The EST library will provide useful information for transcriptional analyses, proteomic analyses, and gene functional analyses of BPH. Moreover, specific genes for hemimetabolous insects will be identified. The microarray fabricated based on the EST information will be useful for finding genes related to agricultural and biological problems related to this pest.

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

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.

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.

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

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.

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

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.

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...

Library of molecular associations: curating the complex molecular basis of liver diseases.

PubMed

Buchkremer, Stefan; Hendel, Jasmin; Krupp, Markus; Weinmann, Arndt; Schlamp, Kai; Maass, Thorsten; Staib, Frank; Galle, Peter R; Teufel, Andreas

2010-03-20

Systems biology approaches offer novel insights into the development of chronic liver diseases. Current genomic databases supporting systems biology analyses are mostly based on microarray data. Although these data often cover genome wide expression, the validity of single microarray experiments remains questionable. However, for systems biology approaches addressing the interactions of molecular networks comprehensive but also highly validated data are necessary. We have therefore generated the first comprehensive database for published molecular associations in human liver diseases. It is based on PubMed published abstracts and aimed to close the gap between genome wide coverage of low validity from microarray data and individual highly validated data from PubMed. After an initial text mining process, the extracted abstracts were all manually validated to confirm content and potential genetic associations and may therefore be highly trusted. All data were stored in a publicly available database, Library of Molecular Associations http://www.medicalgenomics.org/databases/loma/news, currently holding approximately 1260 confirmed molecular associations for chronic liver diseases such as HCC, CCC, liver fibrosis, NASH/fatty liver disease, AIH, PBC, and PSC. We furthermore transformed these data into a powerful resource for molecular liver research by connecting them to multiple biomedical information resources. Together, this database is the first available database providing a comprehensive view and analysis options for published molecular associations on multiple liver diseases.

Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord.

PubMed

Tamplin, Owen J; Cox, Brian J; Rossant, Janet

2011-12-15

The node and notochord are key tissues required for patterning of the vertebrate body plan. Understanding the gene regulatory network that drives their formation and function is therefore important. Foxa2 is a key transcription factor at the top of this genetic hierarchy and finding its targets will help us to better understand node and notochord development. We performed an extensive microarray-based gene expression screen using sorted embryonic notochord cells to identify early notochord-enriched genes. We validated their specificity to the node and notochord by whole mount in situ hybridization. This provides the largest available resource of notochord-expressed genes, and therefore candidate Foxa2 target genes in the notochord. Using existing Foxa2 ChIP-seq data from adult liver, we were able to identify a set of genes expressed in the notochord that had associated regions of Foxa2-bound chromatin. Given that Foxa2 is a pioneer transcription factor, we reasoned that these sites might represent notochord-specific enhancers. Candidate Foxa2-bound regions were tested for notochord specific enhancer function in a zebrafish reporter assay and 7 novel notochord enhancers were identified. Importantly, sequence conservation or predictive models could not have readily identified these regions. Mutation of putative Foxa2 binding elements in two of these novel enhancers abrogated reporter expression and confirmed their Foxa2 dependence. The combination of highly specific gene expression profiling and genome-wide ChIP analysis is a powerful means of understanding developmental pathways, even for small cell populations such as the notochord. Copyright © 2011 Elsevier Inc. 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.

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

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.

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

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

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.

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.

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.

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.

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.

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.

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

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

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins.

PubMed

Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

2007-12-21

Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

PubMed Central

Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

2007-01-01

Background Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences. PMID:18154678

A universal reference sample derived from clone vector for improved detection of differential gene expression

PubMed Central

Khan, Rishi L; Gonye, Gregory E; Gao, Guang; Schwaber, James S

2006-01-01

Background Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs™. Results We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. Conclusion Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes. PMID:16677381

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

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.

The MGED ontology: a framework for describing functional genomics experiments.

PubMed

Stoeckert, Christian J; Parkinson, Helen

2003-01-01

The Microarray Gene Expression Data (MGED) society was formed with an initial focus on experiments involving microarray technology. Despite the diversity of applications, there are common concepts used and a common need to capture experimental information in a standardized manner. In building the MGED ontology, it was recognized that it would be impractical to cover all the different types of experiments on all the different types of organisms by listing and defining all the types of organisms and their properties. Our solution was to create a framework for describing microarray experiments with an initial focus on the biological sample and its manipulation. For concepts that are common for many species, we could provide a manageable listing of controlled terms. For concepts that are species-specific or whose values cannot be readily listed, we created an 'OntologyEntry' concept that referenced an external resource. The MGED ontology is a work in progress that needs additional instances and particularly needs constraints to be added. The ontology currently covers the experimental sample and design, and we have begun capturing aspects of the microarrays themselves as well. The primary application of the ontology will be to develop forms for entering information into databases, and consequently allowing queries, taking advantage of the structure provided by the ontology. The application of an ontology of experimental conditions extends beyond microarray experiments and, as the scope of MGED includes other aspects of functional genomics, so too will the MGED ontology.

DNA microarray technology in nutraceutical and food safety.

PubMed

Liu-Stratton, Yiwen; Roy, Sashwati; Sen, Chandan K

2004-04-15

The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

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.

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

Application of microarray analysis on computer cluster and cloud platforms.

PubMed

Bernau, C; Boulesteix, A-L; Knaus, J

2013-01-01

Analysis of recent high-dimensional biological data tends to be computationally intensive as many common approaches such as resampling or permutation tests require the basic statistical analysis to be repeated many times. A crucial advantage of these methods is that they can be easily parallelized due to the computational independence of the resampling or permutation iterations, which has induced many statistics departments to establish their own computer clusters. An alternative is to rent computing resources in the cloud, e.g. at Amazon Web Services. In this article we analyze whether a selection of statistical projects, recently implemented at our department, can be efficiently realized on these cloud resources. Moreover, we illustrate an opportunity to combine computer cluster and cloud resources. In order to compare the efficiency of computer cluster and cloud implementations and their respective parallelizations we use microarray analysis procedures and compare their runtimes on the different platforms. Amazon Web Services provide various instance types which meet the particular needs of the different statistical projects we analyzed in this paper. Moreover, the network capacity is sufficient and the parallelization is comparable in efficiency to standard computer cluster implementations. Our results suggest that many statistical projects can be efficiently realized on cloud resources. It is important to mention, however, that workflows can change substantially as a result of a shift from computer cluster to cloud computing.

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.

Arabidopsis Gene Family Profiler (aGFP)--user-oriented transcriptomic database with easy-to-use graphic interface.

PubMed

Dupl'áková, Nikoleta; Renák, David; Hovanec, Patrik; Honysová, Barbora; Twell, David; Honys, David

2007-07-23

Microarray technologies now belong to the standard functional genomics toolbox and have undergone massive development leading to increased genome coverage, accuracy and reliability. The number of experiments exploiting microarray technology has markedly increased in recent years. In parallel with the rapid accumulation of transcriptomic data, on-line analysis tools are being introduced to simplify their use. Global statistical data analysis methods contribute to the development of overall concepts about gene expression patterns and to query and compose working hypotheses. More recently, these applications are being supplemented with more specialized products offering visualization and specific data mining tools. We present a curated gene family-oriented gene expression database, Arabidopsis Gene Family Profiler (aGFP; http://agfp.ueb.cas.cz), which gives the user access to a large collection of normalised Affymetrix ATH1 microarray datasets. The database currently contains NASC Array and AtGenExpress transcriptomic datasets for various tissues at different developmental stages of wild type plants gathered from nearly 350 gene chips. The Arabidopsis GFP database has been designed as an easy-to-use tool for users needing an easily accessible resource for expression data of single genes, pre-defined gene families or custom gene sets, with the further possibility of keyword search. Arabidopsis Gene Family Profiler presents a user-friendly web interface using both graphic and text output. Data are stored at the MySQL server and individual queries are created in PHP script. The most distinguishable features of Arabidopsis Gene Family Profiler database are: 1) the presentation of normalized datasets (Affymetrix MAS algorithm and calculation of model-based gene-expression values based on the Perfect Match-only model); 2) the choice between two different normalization algorithms (Affymetrix MAS4 or MAS5 algorithms); 3) an intuitive interface; 4) an interactive "virtual plant" visualizing the spatial and developmental expression profiles of both gene families and individual genes. Arabidopsis GFP gives users the possibility to analyze current Arabidopsis developmental transcriptomic data starting with simple global queries that can be expanded and further refined to visualize comparative and highly selective gene expression profiles.

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.

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).

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.

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

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

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.

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

BμG@Sbase—a microbial gene expression and comparative genomic database

PubMed Central

Witney, Adam A.; Waldron, Denise E.; Brooks, Lucy A.; Tyler, Richard H.; Withers, Michael; Stoker, Neil G.; Wren, Brendan W.; Butcher, Philip D.; Hinds, Jason

2012-01-01

The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BμG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BμG@Sbase, a microbial gene expression and comparative genomic database. BμG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future. PMID:21948792

BμG@Sbase--a microbial gene expression and comparative genomic database.

PubMed

Witney, Adam A; Waldron, Denise E; Brooks, Lucy A; Tyler, Richard H; Withers, Michael; Stoker, Neil G; Wren, Brendan W; Butcher, Philip D; Hinds, Jason

2012-01-01

The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BμG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BμG@Sbase, a microbial gene expression and comparative genomic database. BμG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future.

Curation of microarray oligonucleotides and corresponding ESTs/cDNAs used for gene expression analysis in zebra finches.

PubMed

Lovell, Peter V; Huizinga, Nicole A; Getachew, Abel; Mees, Brianna; Friedrich, Samantha R; Wirthlin, Morgan; Mello, Claudio V

2018-05-18

Zebra finches are a major model organism for investigating mechanisms of vocal learning, a trait that enables spoken language in humans. The development of cDNA collections with expressed sequence tags (ESTs) and microarrays has allowed for extensive molecular characterizations of circuitry underlying vocal learning and production. However, poor database curation can lead to errors in transcriptome and bioinformatics analyses, limiting the impact of these resources. Here we used genomic alignments and synteny analysis for orthology verification to curate and reannotate ~ 35% of the oligonucleotides and corresponding ESTs/cDNAs that make-up Agilent microarrays for gene expression analysis in finches. We found that: (1) 5475 out of 43,084 oligos (a) failed to align to the zebra finch genome, (b) aligned to multiple loci, or (c) aligned to Chr_un only, and thus need to be flagged until a better genome assembly is available, or (d) reflect cloning artifacts; (2) Out of 9635 valid oligos examined further, 3120 were incorrectly named, including 1533 with no known orthologs; and (3) 2635 oligos required name update. The resulting curated dataset provides a reference for correcting gene identification errors in previous finch microarrays studies, and avoiding such errors in future studies.

Genomics of compositae weeds: EST libraries, microarrays, and evidence of introgression

USDA-ARS?s Scientific Manuscript database

• Premise of Study: Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we report on the development of genomic tools and resources for 11 weeds from the Compositae family that can serve ...

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.

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

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.

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.

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.

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.

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

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.

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

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.

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

Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: A genomic resource for studying agricultural pests

PubMed Central

Noda, Hiroaki; Kawai, Sawako; Koizumi, Yoko; Matsui, Kageaki; Zhang, Qiang; Furukawa, Shigetoyo; Shimomura, Michihiko; Mita, Kazuei

2008-01-01

Background The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is a serious insect pests of rice plants. Major means of BPH control are application of agricultural chemicals and cultivation of BPH resistant rice varieties. Nevertheless, BPH strains that are resistant to agricultural chemicals have developed, and BPH strains have appeared that are virulent against the resistant rice varieties. Expressed sequence tag (EST) analysis and related applications are useful to elucidate the mechanisms of resistance and virulence and to reveal physiological aspects of this non-model insect, with its poorly understood genetic background. Results More than 37,000 high-quality ESTs, excluding sequences of mitochondrial genome, microbial genomes, and rDNA, have been produced from 18 libraries of various BPH tissues and stages. About 10,200 clusters have been made from whole EST sequences, with average EST size of 627 bp. Among the top ten most abundantly expressed genes, three are unique and show no homology in BLAST searches. The actin gene was highly expressed in BPH, especially in the thorax. Tissue-specifically expressed genes were extracted based on the expression frequency among the libraries. An EST database is available at our web site. Conclusion The EST library will provide useful information for transcriptional analyses, proteomic analyses, and gene functional analyses of BPH. Moreover, specific genes for hemimetabolous insects will be identified. The microarray fabricated based on the EST information will be useful for finding genes related to agricultural and biological problems related to this pest. PMID:18315884

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

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...

Mining the archives: a cross-platform analysis of gene ...

EPA Pesticide Factsheets

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for genomic research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation of nucleic acids. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues using two DNA microarray protocols and two whole transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other three methods by having the highest correlations of differentially expressed genes (DEGs) and best overlap of pathways between FRO and FFPE groups. We next tested the effect of sample time in formalin (18 hours or 3 weeks) on gene expression profiles. Hierarchical clustering of the datasets indicated that test article treatment, and not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18-hour and 3-week FFPE samples compared to FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of age in FFPE block on genomic profiles. RNA-seq analysis of 8-, 19-, and 26-year-old control blocks using the ribo-depletion protocol resulted in comparable quality metrics, inc

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.

Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

NASA Astrophysics Data System (ADS)

Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

2011-06-01

Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

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

Within and between Whorls: Comparative Transcriptional Profiling of Aquilegia and Arabidopsis

PubMed Central

Voelckel, Claudia; Borevitz, Justin O.; Kramer, Elena M.; Hodges, Scott A.

2010-01-01

Background The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels. Methodology/Principal Findings We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource). Conclusions/Significance Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology. PMID:20352114

Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.

PubMed

Son, M-Y; Sim, H; Son, Y S; Jung, K B; Lee, M-O; Oh, J-H; Chung, S-K; Jung, C-R; Kim, J

2017-12-01

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids. © 2017 British Neuropathological Society.

Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia.

PubMed

Segara, Davendra; Biankin, Andrew V; Kench, James G; Langusch, Catherine C; Dawson, Amanda C; Skalicky, David A; Gotley, David C; Coleman, Maxwell J; Sutherland, Robert L; Henshall, Susan M

2005-05-01

Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARalpha, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and PanIN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early PanIN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

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.

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.

Computational, Integrative, and Comparative Methods for the Elucidation of Genetic Coexpression Networks

DOE PAGES

Baldwin, Nicole E.; Chesler, Elissa J.; Kirov, Stefan; ...

2005-01-01

Gene expression microarray data can be used for the assembly of genetic coexpression network graphs. Using mRNA samples obtained from recombinant inbred Mus musculus strains, it is possible to integrate allelic variation with molecular and higher-order phenotypes. The depth of quantitative genetic analysis of microarray data can be vastly enhanced utilizing this mouse resource in combination with powerful computational algorithms, platforms, and data repositories. The resulting network graphs transect many levels of biological scale. This approach is illustrated with the extraction of cliques of putatively co-regulated genes and their annotation using gene ontology analysis and cis -regulatory element discovery. Themore » causal basis for co-regulation is detected through the use of quantitative trait locus mapping.« less

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.

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

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.

MiMiR – an integrated platform for microarray data sharing, mining and analysis

PubMed Central

Tomlinson, Chris; Thimma, Manjula; Alexandrakis, Stelios; Castillo, Tito; Dennis, Jayne L; Brooks, Anthony; Bradley, Thomas; Turnbull, Carly; Blaveri, Ekaterini; Barton, Geraint; Chiba, Norie; Maratou, Klio; Soutter, Pat; Aitman, Tim; Game, Laurence

2008-01-01

Background Despite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Microarray data Mining Resource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data. Results A user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package. Conclusion The new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centre's large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies. PMID:18801157

MiMiR--an integrated platform for microarray data sharing, mining and analysis.

PubMed

Tomlinson, Chris; Thimma, Manjula; Alexandrakis, Stelios; Castillo, Tito; Dennis, Jayne L; Brooks, Anthony; Bradley, Thomas; Turnbull, Carly; Blaveri, Ekaterini; Barton, Geraint; Chiba, Norie; Maratou, Klio; Soutter, Pat; Aitman, Tim; Game, Laurence

2008-09-18

Despite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Microarray data Mining Resource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data. A user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package. The new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centre's large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies.

VectorBase: a data resource for invertebrate vector genomics

PubMed Central

Lawson, Daniel; Arensburger, Peter; Atkinson, Peter; Besansky, Nora J.; Bruggner, Robert V.; Butler, Ryan; Campbell, Kathryn S.; Christophides, George K.; Christley, Scott; Dialynas, Emmanuel; Hammond, Martin; Hill, Catherine A.; Konopinski, Nathan; Lobo, Neil F.; MacCallum, Robert M.; Madey, Greg; Megy, Karine; Meyer, Jason; Redmond, Seth; Severson, David W.; Stinson, Eric O.; Topalis, Pantelis; Birney, Ewan; Gelbart, William M.; Kafatos, Fotis C.; Louis, Christos; Collins, Frank H.

2009-01-01

VectorBase (http://www.vectorbase.org) is an NIAID-funded Bioinformatic Resource Center focused on invertebrate vectors of human pathogens. VectorBase annotates and curates vector genomes providing a web accessible integrated resource for the research community. Currently, VectorBase contains genome information for three mosquito species: Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus, a body louse Pediculus humanus and a tick species Ixodes scapularis. Since our last report VectorBase has initiated a community annotation system, a microarray and gene expression repository and controlled vocabularies for anatomy and insecticide resistance. We have continued to develop both the software infrastructure and tools for interrogating the stored data. PMID:19028744

The emergence and diffusion of DNA microarray technology.

PubMed

Lenoir, Tim; Giannella, Eric

2006-08-22

The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow--namely; the flow of inventions into industry through the licensing of university-based technologies--has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail. We discuss the role of federal funding of academic research in the microarray field, and the multiple pathways through which federally supported development of commercial microarray technologies have transformed core academic research fields. Our study confirms the picture put forward by several scholars that the open character of networked economies is what makes them truly innovative. In an open system innovations emerge from the network. The emergence and diffusion of microarray technologies we have traced here provides an excellent example of an open system of innovation in action. Whether they originated in a startup company environment that operated like a think-tank, such as Affymax, the research labs of a large firm, such as Agilent, or within a research university, the inventors we have followed drew heavily on knowledge resources from all parts of the network in bringing microarray platforms to light. Federal funding for high-tech startups and new industrial development was important at several phases in the early history of microarrays, and federal funding of academic researchers using microarrays was fundamental to transforming the research agendas of several fields within academe. The typical story told about the role of federal funding emphasizes the spillovers from federally funded academic research to industry. Our study shows that the knowledge spillovers worked both ways, with federal funding of non-university research providing the impetus for reshaping the research agendas of several academic fields.

The emergence and diffusion of DNA microarray technology

PubMed Central

Lenoir, Tim; Giannella, Eric

2006-01-01

The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow – namely; the flow of inventions into industry through the licensing of university-based technologies – has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail. We discuss the role of federal funding of academic research in the microarray field, and the multiple pathways through which federally supported development of commercial microarray technologies have transformed core academic research fields. Our study confirms the picture put forward by several scholars that the open character of networked economies is what makes them truly innovative. In an open system innovations emerge from the network. The emergence and diffusion of microarray technologies we have traced here provides an excellent example of an open system of innovation in action. Whether they originated in a startup company environment that operated like a think-tank, such as Affymax, the research labs of a large firm, such as Agilent, or within a research university, the inventors we have followed drew heavily on knowledge resources from all parts of the network in bringing microarray platforms to light. Federal funding for high-tech startups and new industrial development was important at several phases in the early history of microarrays, and federal funding of academic researchers using microarrays was fundamental to transforming the research agendas of several fields within academe. The typical story told about the role of federal funding emphasizes the spillovers from federally funded academic research to industry. Our study shows that the knowledge spillovers worked both ways, with federal funding of non-university research providing the impetus for reshaping the research agendas of several academic fields. PMID:16925816

A low density microarray method for the identification of human papillomavirus type 18 variants.

PubMed

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C

2013-09-26

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

PubMed Central

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

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

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.

The contribution of the DNA microarray technology to gene expression profiling in Leishmania spp.: a retrospective.

PubMed

Alonso, Ana; Larraga, Vicente; Alcolea, Pedro J

2018-05-07

The first genome project of any living organism excluding viruses, the gammaproteobacteria Haemophilus influenzae, was completed in 1995. Until the last decade, genome sequencing was very tedious because genome survey sequences (GSS) and/or expressed sequence tags (ESTs) belonging to plasmid, cosmid and artificial chromosome genome libraries had to be sequenced and assembled in silico. Nowadays, no genome is completely assembled actually, because gaps and unassembled contigs are always remaining. However, most represent the whole genome of the organism of origin from a practical point of view. The first genome sequencing projects of trypanosomatid parasites were completed in 2005 following those strategies, and belong to Leishmania major, Trypanosoma cruzi and T. brucei. The functional genomics era rapidly developed on the basis of the microarray technology and has been evolving. In the case of the genus Leishmania, substantial biological information about differentiation in the digenetic life cycle of the parasite has been obtained. Later on, next generation sequencing has revolutionized genome sequencing and functional genomics, leading to more sensitive, accurate results by using much less resources. This new technology is more advantageous, but does not invalidate microarray results. In fact, promising vaccine candidates and drug targets have been found on the basis of microarray-based screening and preliminary proof-of-concept tests. Copyright © 2018. Published by Elsevier B.V.

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

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

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

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.

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

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

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.

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.

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.

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

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

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.

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.

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

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.

Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids

PubMed Central

Srinivasan, Mythily; Sedmak, Daniel; Jewell, Scott

2002-01-01

Clinical and molecular medicines are undergoing a revolution based on the accelerated advances in biotechnology such as DNA microarrays and proteomics. Answers to fundamental questions such as how does the DNA sequence differ between individuals and what makes one individual more prone for a certain disease are eagerly being sought in this postgenomic era. Several government and nonprofit organizations provide the researchers access to human tissues for molecular studies. The tissues procured by the different organizations may differ with respect to fixation and processing parameters that may affect significantly the molecular profile of the tissues. It is imperative that a prospective investigator be aware of the potential contributing factors before designing a project. The purpose of this review is to provide an overview of the methods of human tissue acquisition, fixation, and preservation. In addition, the parameters of procurement and fixation that affect the quality of the tissues at the molecular level are discussed. PMID:12466110

Hyperspectral microscopic analysis of normal, benign and carcinoma microarray tissue sections

NASA Astrophysics Data System (ADS)

Maggioni, Mauro; Davis, Gustave L.; Warner, Frederick J.; Geshwind, Frank B.; Coppi, Andreas C.; DeVerse, Richard A.; Coifman, Ronald R.

2006-02-01

We apply a unique micro-optoelectromechanical tuned light source and new algorithms to the hyper-spectral microscopic analysis of human colon biopsies. The tuned light prototype (Plain Sight Systems Inc.) transmits any combination of light frequencies, range 440nm 700nm, trans-illuminating H and E stained tissue sections of normal (N), benign adenoma (B) and malignant carcinoma (M) colon biopsies, through a Nikon Biophot microscope. Hyper-spectral photomicrographs, randomly collected 400X magnication, are obtained with a CCD camera (Sensovation) from 59 different patient biopsies (20 N, 19 B, 20 M) mounted as a microarray on a single glass slide. The spectra of each pixel are normalized and analyzed to discriminate among tissue features: gland nuclei, gland cytoplasm and lamina propria/lumens. Spectral features permit the automatic extraction of 3298 nuclei with classification as N, B or M. When nuclei are extracted from each of the 59 biopsies the average classification among N, B and M nuclei is 97.1%; classification of the biopsies, based on the average nuclei classification, is 100%. However, when the nuclei are extracted from a subset of biopsies, and the prediction is made on nuclei in the remaining biopsies, there is a marked decrement in performance to 60% across the 3 classes. Similarly the biopsy classification drops to 54%. In spite of these classification differences, which we believe are due to instrument and biopsy normalization issues, hyper-spectral analysis has the potential to achieve diagnostic efficiency needed for objective microscopic diagnosis.

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

Single cell gene expression profiling in Alzheimer's disease.

PubMed

Ginsberg, Stephen D; Che, Shaoli; Counts, Scott E; Mufson, Elliott J

2006-07-01

Development and implementation of microarray techniques to quantify expression levels of dozens to hundreds to thousands of transcripts simultaneously within select tissue samples from normal control subjects and neurodegenerative diseased brains has enabled scientists to create molecular fingerprints of vulnerable neuronal populations in Alzheimer's disease (AD) and related disorders. A goal is to sample gene expression from homogeneous cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and nonneuronal cells. The precise resolution afforded by single cell and population cell RNA analysis in combination with microarrays and real-time quantitative polymerase chain reaction (qPCR)-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease progression. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models of neurodegeneration as well as postmortem human brain tissues. Gene expression analysis in postmortem AD brain regions including the hippocampal formation and neocortex reveals selectively vulnerable cell types share putative pathogenetic alterations in common classes of transcripts, for example, markers of glutamatergic neurotransmission, synaptic-related markers, protein phosphatases and kinases, and neurotrophins/neurotrophin receptors. Expression profiles of vulnerable regions and neurons may reveal important clues toward the understanding of the molecular pathogenesis of various neurological diseases and aid in identifying rational targets toward pharmacotherapeutic interventions for progressive, late-onset neurodegenerative disorders such as mild cognitive impairment (MCI) and AD.

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.

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

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

PubMed Central

Holman, Edna C.; Campbell, Leah J.; Hines, John; Crews, Craig M.

2012-01-01

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes. PMID:23028429

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.

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.

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.

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

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.

miR-125b-1 and miR-378a are predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer.

PubMed

Tanaka, Hironori; Hazama, Shoichi; Iida, Michihisa; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Nakajima, Masao; Tokumitsu, Yukio; Kanekiyo, Shinsuke; Shindo, Yoshitaro; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Yamamoto, Shigeru; Yoshino, Shigefumi; Ueno, Tomio; Hamamoto, Yoshihiko; Fujita, Yusuke; Tanaka, Hiroaki; Tahara, Ko; Shimizu, Ryoichi; Okuno, Kiyotaka; Fujita, Koji; Kuroda, Masahiko; Nakamura, Yusuke; Nagano, Hiroaki

2017-11-01

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in a phase I study, in which peptide vaccines alone were given. Candidate miRNAs were selected by comparison of the better prognosis group with the poorer prognosis group. Next, we conducted microarray analysis of cancer tissues in a phase II study, in which peptide vaccines combined with chemotherapy were given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse-transcription PCR analysis of phase II cases, separating cancer tissues into cancer cells and stromal tissue using laser capture microdissection. Treatment effect in relation to overall survival (OS) and miRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR-125b-1 in cancer cells (P = 0.040), and miR-378a in both cancer cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that expression of miR-378a in stromal cells was the best among the three predictors (HR, 2.730; 95% CI, 1.027-7.585; P = 0.044). In conclusion, miR-125b-1 and miR-378a expression might be considered as novel biomarkers to predict the efficacy of vaccine treatment against colorectal cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Aqueous extracts and polysaccharides from Marshmallow roots (Althea officinalis L.): cellular internalisation and stimulation of cell physiology of human epithelial cells in vitro.

PubMed

Deters, Alexandra; Zippel, Janina; Hellenbrand, Nils; Pappai, Dirk; Possemeyer, Cathleen; Hensel, Andreas

2010-01-08

Aqueous extracts from the roots of Althea officinalis L. (Malvaceae) are widely used for treatment of irritated mucosa. The clinical proven effects are related to the presence of bioadhesive and mucilaginous polysaccharides from the rhamnogalacturonan type, leading to the physical formation of mucin-like on top of the irritated tissues. No data are available if the extracts or the polysaccharides from these extract exert an active influence on mucosal or connective tissue cells, in order to initiated changes in cell physiology, useful for better tissue regeneration. In vitro investigations of aqueous A. officinalis extract AE and raw polysaccharides (RPS) on epithelial KB cells and primary dermal human fibroblasts (pNHF) using WST1 vitality test and BrdU proliferation ELISA. Gene expression analysis by microarray from KB cells. Internalisation studies of polysaccharides were performed by laser scanning microscopy. AE (1, 10 microg/mL) had stimulating effect on cell viability and proliferation of epithelial KB cells. RPS (1, 10 microg/mL) stimulated cell vitality of epithelial cells significantly without triggering the cells into higher proliferation status. Neither AE nor RPS had any effect on fibroblasts. FITC-labeled RPS was shown to be internalised into epithelial cells, but not into fibroblasts. FITC-RPS was shown to form bioadhesive layers on the cell surface of dermal fibroblasts. Microarray analysis indicated an up-regulation of genes related to cell adhesion proteins, growth regulators, extracellular matrix, cytokine release and apoptosis. Aqueous extracts and polysaccharides from the roots of A. officinalis are effective stimulators of cell physiology of epithelial cells which can prove the traditional use of Marshmallow preparations for treatment of irritated mucous membranes within tissue regeneration. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

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

High Resolution Analysis of Copy Number Mutation in Breast Cancer

DTIC Science & Technology

2005-05-01

tissues and Epstein - Barr sentations and arrays of Hind III probes additional CNPs, as would an increase in the virus -immortalized lymphoblastoid cell...software and laboratory procedures for the design of inter-phase FISH primers. We have also made progress in developing database and data processing...Cancer progression often involves alterations in DNA copy number. Newly developed microarray technologies enable simultane- ous measurement of copy

Global gene expression profiles of Phytophthora ramorum strain pr102 in response to plant host and tissue differentiation

Treesearch

Caroline M. Press; Niklaus J. Grunwald

2008-01-01

The release of the draft genome sequence of P. ramorum strain Pr102, enabled the construction of an oligonucleotide microarray of the entire genome of Pr102. The array contains 344,680 features (oligos) that represent the transcriptome of Pr102. P. ramorum RNA was extracted from mycelium and sporangia and used to compare gene...

Identification of Prostate Cancer Prognostic Markers

DTIC Science & Technology

2015-10-01

downregulation of GABARAPL2, a gene located in a chromosomal region deleted in PCa metastases, showed increase in autophagy in a PCa cell line and reduced...alteration, chromosome gain and deletion, fluorescence in situ hybridization (FISH), prognostic markers, biomarkers, tissue microarrays, autophagy 16...TMA), colony formation assay, cell growth, autophagy . 3. ACCOMPLISHMENTS: What were the major goals of the project? The hypothesis of the project is

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.

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.

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

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.

Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

PubMed Central

LING, SHIZHANG; RETTIG, ELENI M.; TAN, MARIETTA; CHANG, XIAOFEI; WANG, ZHIMING; BRAIT, MARIANA; BISHOP, JUSTIN A.; FERTIG, ELANA J.; CONSIDINE, MICHAEL; WICK, MICHAEL J.; HA, PATRICK K.

2016-01-01

Salivary gland adenoid cystic carcinoma (ACC) is a rare head and neck malignancy without molecular biomarkers that can be used to predict the chemotherapeutic response or prognosis of ACC. The regulation of gene expression of oncogenes and tumor suppressor genes (TSGs) through DNA promoter methylation may play a role in the carcinogenesis of ACC. To identify differentially methylated genes in ACC, a global demethylating agent, 5-aza-2′-deoxycytidine (5-AZA) was utilized to unmask putative TSG silencing in ACC xenograft models in mice. Fresh xenografts were passaged, implanted in triplicate in mice that were treated with 5-AZA daily for 28 days. These xenografts were then evaluated for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation27 BeadChip array. Validation of the 32 candidate genes was performed by bisulfite sequencing (BS-seq) in a separate cohort of 6 ACC primary tumors and 6 normal control salivary gland tissues. Hypermethylation was identified in the HCN2 gene promoter in all 6 control tissues, but hypomethylation was found in all 6 ACC tumor tissues. Quantitative validation of HCN2 promoter methylation level in the region detected by BS-seq was performed in a larger cohort of primary tumors (n=32) confirming significant HCN2 hypomethylation in ACCs compared with normal samples (n=10; P=0.04). HCN2 immunohistochemical staining was performed on an ACC tissue microarray. HCN2 staining intensity and H-score, but not percentage of the positively stained cells, were significantly stronger in normal tissues than those of ACC tissues. With our novel screening and sequencing methods, we identified several gene candidates that were methylated. The most significant of these genes, HCN2, was actually hypomethylated in tumors. However, promoter methylation status does not appear to be a major determinant of HCN2 expression in normal and ACC tissues. HCN2 hypomethylation is a biomarker of ACC and may play an important role in the carcinogenesis of ACC. PMID:27212063

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

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

Lambros, Maria Polikandritou, E-mail: mlambros@westernu.edu; Parsa, Cyrus; Mulamalla, HariChandana

2011-02-04

Research highlights: {yields} We irradiated a 3-D human oral cell culture of keratinocytes and fibroblasts with 12 and 2 Gy. {yields} 6 h after irradiation the histopathology and apoptosis of the 3-D culture were evaluated. Microarrays were used to assess the gene expression in the irradiated 3-D tissue. {yields} 12 Gy induced significant histopathologic changes and cellular apoptosis. {yields} 12 Gy significantly affected genes of the NF-kB pathway, inflammatory cytokines and DAMPs. -- Abstract: Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinelymore » be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.« less

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome

PubMed Central

2014-01-01

Background Long noncoding RNAs (lncRNAs) constitute a major, but poorly characterized part of human transcriptome. Recent evidence indicates that many lncRNAs are involved in cancer and can be used as predictive and prognostic biomarkers. Significant fraction of lncRNAs is represented on widely used microarray platforms, however they have usually been ignored in cancer studies. Results We developed a computational pipeline to annotate lncRNAs on popular Affymetrix U133 microarrays, creating a resource allowing measurement of expression of 1581 lncRNAs. This resource can be utilized to interrogate existing microarray datasets for various lncRNA studies. We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length. Remarkably, these three classes of lncRNAs were co-localized with protein coding genes exhibiting distinct gene ontology groups. This annotation was applied to microarray analysis which identified a 159 lncRNA signature that discriminates between localized and metastatic stages of neuroblastoma. Analysis of an independent patient cohort revealed that this signature differentiates also relapsing from non-relapsing primary tumors. This is the first example of the signature developed via the analysis of expression of lncRNAs solely. One of these lncRNAs, termed HOXD-AS1, is encoded in HOXD cluster. HOXD-AS1 is evolutionary conserved among hominids and has all bona fide features of a gene. Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation. Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer. Conclusions Our findings greatly extend the number of noncoding RNAs functionally implicated in tumor development and patient treatment and highlight their role as potential prognostic biomarkers of neuroblastomas. PMID:25522241

The laboratory-clinician team: a professional call to action to improve communication and collaboration for optimal patient care in chromosomal microarray testing.

PubMed

Wain, Karen E; Riggs, Erin; Hanson, Karen; Savage, Melissa; Riethmaier, Darlene; Muirhead, Andrea; Mitchell, Elyse; Packard, Bethanny Smith; Faucett, W Andrew

2012-10-01

The International Standards for Cytogenomic Arrays (ISCA) Consortium is a worldwide collaborative effort dedicated to optimizing patient care by improving the quality of chromosomal microarray testing. The primary effort of the ISCA Consortium has been the development of a database of copy number variants (CNVs) identified during the course of clinical microarray testing. This database is a powerful resource for clinicians, laboratories, and researchers, and can be utilized for a variety of applications, such as facilitating standardized interpretations of certain CNVs across laboratories or providing phenotypic information for counseling purposes when published data is sparse. A recognized limitation to the clinical utility of this database, however, is the quality of clinical information available for each patient. Clinical genetic counselors are uniquely suited to facilitate the communication of this information to the laboratory by virtue of their existing clinical responsibilities, case management skills, and appreciation of the evolving nature of scientific knowledge. We intend to highlight the critical role that genetic counselors play in ensuring optimal patient care through contributing to the clinical utility of the ISCA Consortium's database, as well as the quality of individual patient microarray reports provided by contributing laboratories. Current tools, paper and electronic forms, created to maximize this collaboration are shared. In addition to making a professional commitment to providing complete clinical information, genetic counselors are invited to become ISCA members and to become involved in the discussions and initiatives within the Consortium.

Power enhancement via multivariate outlier testing with gene expression arrays.

PubMed

Asare, Adam L; Gao, Zhong; Carey, Vincent J; Wang, Richard; Seyfert-Margolis, Vicki

2009-01-01

As the use of microarrays in human studies continues to increase, stringent quality assurance is necessary to ensure accurate experimental interpretation. We present a formal approach for microarray quality assessment that is based on dimension reduction of established measures of signal and noise components of expression followed by parametric multivariate outlier testing. We applied our approach to several data resources. First, as a negative control, we found that the Affymetrix and Illumina contributions to MAQC data were free from outliers at a nominal outlier flagging rate of alpha=0.01. Second, we created a tunable framework for artificially corrupting intensity data from the Affymetrix Latin Square spike-in experiment to allow investigation of sensitivity and specificity of quality assurance (QA) criteria. Third, we applied the procedure to 507 Affymetrix microarray GeneChips processed with RNA from human peripheral blood samples. We show that exclusion of arrays by this approach substantially increases inferential power, or the ability to detect differential expression, in large clinical studies. http://bioconductor.org/packages/2.3/bioc/html/arrayMvout.html and http://bioconductor.org/packages/2.3/bioc/html/affyContam.html affyContam (credentials: readonly/readonly)

A Discovery Resource of Rare Copy Number Variations in Individuals with Autism Spectrum Disorder

PubMed Central

Prasad, Aparna; Merico, Daniele; Thiruvahindrapuram, Bhooma; Wei, John; Lionel, Anath C.; Sato, Daisuke; Rickaby, Jessica; Lu, Chao; Szatmari, Peter; Roberts, Wendy; Fernandez, Bridget A.; Marshall, Christian R.; Hatchwell, Eli; Eis, Peggy S.; Scherer, Stephen W.

2012-01-01

The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection. PMID:23275889

Microscale Bioadhesive Hydrogel Arrays for Cell Engineering Applications.

PubMed

Patel, Ravi Ghanshyam; Purwada, Alberto; Cerchietti, Leandro; Inghirami, Giorgio; Melnick, Ari; Gaharwar, Akhilesh K; Singh, Ankur

2014-09-01

Bioengineered hydrogels have been explored in cell and tissue engineering applications to support cell growth and modulate its behavior. A rationally designed scaffold should allow for encapsulated cells to survive, adhere, proliferate, remodel the niche, and can be used for controlled delivery of biomolecules. Here we report a microarray of composite bioadhesive microgels with modular dimensions, tunable mechanical properties and bulk modified adhesive biomolecule composition. Composite bioadhesive microgels of maleimide functionalized polyethylene glycol (PEG-MAL) with interpenetrating network (IPN) of gelatin ionically cross-linked with silicate nanoparticles were engineered by integrating microfabrication with Michael-type addition chemistry and ionic gelation. By encapsulating clinically relevant anchorage-dependent cervical cancer cells and suspension leukemia cells as cell culture models in these composite microgels, we demonstrate enhanced cell spreading, survival, and metabolic activity compared to control gels. The composite bioadhesive hydrogels represent a platform that could be used to study independent effect of stiffness and adhesive ligand density on cell survival and function. We envision that such microarrays of cell adhesive microenvironments, which do not require harsh chemical and UV crosslinking conditions, will provide a more efficacious cell culture platform that can be used to study cell behavior and survival, function as building blocks to fabricate 3D tissue structures, cell delivery systems, and high throughput drug screening devices.

Microscale Bioadhesive Hydrogel Arrays for Cell Engineering Applications

PubMed Central

PATEL, RAVI GHANSHYAM; PURWADA, ALBERTO; CERCHIETTI, LEANDRO; INGHIRAMI, GIORGIO; MELNICK, ARI; GAHARWAR, AKHILESH K.; SINGH, ANKUR

2014-01-01

Bioengineered hydrogels have been explored in cell and tissue engineering applications to support cell growth and modulate its behavior. A rationally designed scaffold should allow for encapsulated cells to survive, adhere, proliferate, remodel the niche, and can be used for controlled delivery of biomolecules. Here we report a microarray of composite bioadhesive microgels with modular dimensions, tunable mechanical properties and bulk modified adhesive biomolecule composition. Composite bioadhesive microgels of maleimide functionalized polyethylene glycol (PEG-MAL) with interpenetrating network (IPN) of gelatin ionically cross-linked with silicate nanoparticles were engineered by integrating microfabrication with Michael-type addition chemistry and ionic gelation. By encapsulating clinically relevant anchorage-dependent cervical cancer cells and suspension leukemia cells as cell culture models in these composite microgels, we demonstrate enhanced cell spreading, survival, and metabolic activity compared to control gels. The composite bioadhesive hydrogels represent a platform that could be used to study independent effect of stiffness and adhesive ligand density on cell survival and function. We envision that such microarrays of cell adhesive microenvironments, which do not require harsh chemical and UV crosslinking conditions, will provide a more efficacious cell culture platform that can be used to study cell behavior and survival, function as building blocks to fabricate 3D tissue structures, cell delivery systems, and high throughput drug screening devices. PMID:25328548

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.

The analysis of image feature robustness using cometcloud

PubMed Central

Qi, Xin; Kim, Hyunjoo; Xing, Fuyong; Parashar, Manish; Foran, David J.; Yang, Lin

2012-01-01

The robustness of image features is a very important consideration in quantitative image analysis. The objective of this paper is to investigate the robustness of a range of image texture features using hematoxylin stained breast tissue microarray slides which are assessed while simulating different imaging challenges including out of focus, changes in magnification and variations in illumination, noise, compression, distortion, and rotation. We employed five texture analysis methods and tested them while introducing all of the challenges listed above. The texture features that were evaluated include co-occurrence matrix, center-symmetric auto-correlation, texture feature coding method, local binary pattern, and texton. Due to the independence of each transformation and texture descriptor, a network structured combination was proposed and deployed on the Rutgers private cloud. The experiments utilized 20 randomly selected tissue microarray cores. All the combinations of the image transformations and deformations are calculated, and the whole feature extraction procedure was completed in 70 minutes using a cloud equipped with 20 nodes. Center-symmetric auto-correlation outperforms all the other four texture descriptors but also requires the longest computational time. It is roughly 10 times slower than local binary pattern and texton. From a speed perspective, both the local binary pattern and texton features provided excellent performance for classification and content-based image retrieval. PMID:23248759

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.

Root defense analysis against Fusarium oxysporum reveals new regulators to confer resistance

PubMed Central

Chen, Yi Chung; Wong, Chin Lin; Muzzi, Frederico; Vlaardingerbroek, Ido; Kidd, Brendan N.; Schenk, Peer M.

2014-01-01

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including Arabidopsis thaliana. Investigation of the defense response against this pathogen had primarily been conducted using leaf tissue and little was known about the root defense response. In this study, we profiled the expression of root genes after infection with F. oxysporum by microarray analysis. In contrast to the leaf response, root tissue did not show a strong induction of defense-associated gene expression and instead showed a greater proportion of repressed genes. Screening insertion mutants from differentially expressed genes in the microarray uncovered a role for the transcription factor ETHYLENE RESPONSE FACTOR72 (ERF72) in susceptibility to F. oxysporum. Due to the role of ERF72 in suppressing programmed cell death and detoxifying reactive oxygen species (ROS), we examined the pub22/pub23/pub24 U-box type E3 ubiquitin ligase triple mutant which is known to possess enhanced ROS production in response to pathogen challenge. We found that the pub22/23/24 mutant is more resistant to F. oxysporum infection, suggesting that a heightened innate immune response provides protection against F. oxysporum. We conclude that root-mediated defenses against soil-borne pathogens can be provided at multiple levels. PMID:24998294

Image-guided genomic analysis of tissue response to laser-induced thermal stress

NASA Astrophysics Data System (ADS)

Mackanos, Mark A.; Helms, Mike; Kalish, Flora; Contag, Christopher H.

2011-05-01

The cytoprotective response to thermal injury is characterized by transcriptional activation of ``heat shock proteins'' (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.

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.

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.

UniPROBE, update 2011: expanded content and search tools in the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Robasky, Kimberly; Bulyk, Martha L

2011-01-01

The Universal PBM Resource for Oligonucleotide-Binding Evaluation (UniPROBE) database is a centralized repository of information on the DNA-binding preferences of proteins as determined by universal protein-binding microarray (PBM) technology. Each entry for a protein (or protein complex) in UniPROBE provides the quantitative preferences for all possible nucleotide sequence variants ('words') of length k ('k-mers'), as well as position weight matrix (PWM) and graphical sequence logo representations of the k-mer data. In this update, we describe >130% expansion of the database content, incorporation of a protein BLAST (blastp) tool for finding protein sequence matches in UniPROBE, the introduction of UniPROBE accession numbers and additional database enhancements. The UniPROBE database is available at http://uniprobe.org.

Digital microarray analysis for digital artifact genomics

NASA Astrophysics Data System (ADS)

Jaenisch, Holger; Handley, James; Williams, Deborah

2013-06-01

We implement a Spatial Voting (SV) based analogy of microarray analysis for digital gene marker identification in malware code sections. We examine a famous set of malware formally analyzed by Mandiant and code named Advanced Persistent Threat (APT1). APT1 is a Chinese organization formed with specific intent to infiltrate and exploit US resources. Manidant provided a detailed behavior and sting analysis report for the 288 malware samples available. We performed an independent analysis using a new alternative to the traditional dynamic analysis and static analysis we call Spatial Analysis (SA). We perform unsupervised SA on the APT1 originating malware code sections and report our findings. We also show the results of SA performed on some members of the families associated by Manidant. We conclude that SV based SA is a practical fast alternative to dynamics analysis and static analysis.

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.

Identification of embryonic pancreatic genes using Xenopus DNA microarrays.

PubMed

Hayata, Tadayoshi; Blitz, Ira L; Iwata, Nahoko; Cho, Ken W Y

2009-06-01

The pancreas is both an exocrine and endocrine endodermal organ involved in digestion and glucose homeostasis. During embryogenesis, the anlagen of the pancreas arise from dorsal and ventral evaginations of the foregut that later fuse to form a single organ. To better understand the molecular genetics of early pancreas development, we sought to isolate markers that are uniquely expressed in this tissue. Microarray analysis was performed comparing dissected pancreatic buds, liver buds, and the stomach region of tadpole stage Xenopus embryos. A total of 912 genes were found to be differentially expressed between these organs during early stages of organogenesis. K-means clustering analysis predicted 120 of these genes to be specifically enriched in the pancreas. Of these, we report on the novel expression patterns of 24 genes. Our analyses implicate the involvement of previously unsuspected signaling pathways during early pancreas development. Developmental Dynamics 238:1455-1466, 2009. (c) 2009 Wiley-Liss, Inc.

Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

PubMed

Tronser, Tina; Popova, Anna A; Jaggy, Mona; Bastmeyer, Martin; Levkin, Pavel A

2017-12-01

Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of regenerative medicine and tissue engineering. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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

Creation of a digital slide and tissue microarray resource from a multi-institutional predictive toxicology study in the rat: an initial report from the PredTox group.

PubMed

Mulrane, Laoighse; Rexhepaj, Elton; Smart, Valerie; Callanan, John J; Orhan, Diclehan; Eldem, Türkan; Mally, Angela; Schroeder, Susanne; Meyer, Kirstin; Wendt, Maria; O'Shea, Donal; Gallagher, William M

2008-08-01

The widespread use of digital slides has only recently come to the fore with the development of high-throughput scanners and high performance viewing software. This development, along with the optimisation of compression standards and image transfer techniques, has allowed the technology to be used in wide reaching applications including integration of images into hospital information systems and histopathological training, as well as the development of automated image analysis algorithms for prediction of histological aberrations and quantification of immunohistochemical stains. Here, the use of this technology in the creation of a comprehensive library of images of preclinical toxicological relevance is demonstrated. The images, acquired using the Aperio ScanScope CS and XT slide acquisition systems, form part of the ongoing EU FP6 Integrated Project, Innovative Medicines for Europe (InnoMed). In more detail, PredTox (abbreviation for Predictive Toxicology) is a subproject of InnoMed and comprises a consortium of 15 industrial (13 large pharma, 1 technology provider and 1 SME) and three academic partners. The primary aim of this consortium is to assess the value of combining data generated from 'omics technologies (proteomics, transcriptomics, metabolomics) with the results from more conventional toxicology methods, to facilitate further informed decision making in preclinical safety evaluation. A library of 1709 scanned images was created of full-face sections of liver and kidney tissue specimens from male Wistar rats treated with 16 proprietary and reference compounds of known toxicity; additional biological materials from these treated animals were separately used to create 'omics data, that will ultimately be used to populate an integrated toxicological database. In respect to assessment of the digital slides, a web-enabled digital slide management system, Digital SlideServer (DSS), was employed to enable integration of the digital slide content into the 'omics database and to facilitate remote viewing by pathologists connected with the project. DSS also facilitated manual annotation of digital slides by the pathologists, specifically in relation to marking particular lesions of interest. Tissue microarrays (TMAs) were constructed from the specimens for the purpose of creating a repository of tissue from animals used in the study with a view to later-stage biomarker assessment. As the PredTox consortium itself aims to identify new biomarkers of toxicity, these TMAs will be a valuable means of validation. In summary, a large repository of histological images was created enabling the subsequent pathological analysis of samples through remote viewing and, along with the utilisation of TMA technology, will allow the validation of biomarkers identified by the PredTox consortium. The population of the PredTox database with these digitised images represents the creation of the first toxicological database integrating 'omics and preclinical data with histological images.

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.

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.

Characterization of gonadal transcriptomes from the turbot (Scophthalmus maximus).

PubMed

Hu, Yulong; Huang, Meng; Wang, Weiji; Guan, Jiantao; Kong, Jie

2016-01-01

The mechanisms underlying sexual reproduction and sex ratio determination remains unclear in turbot, a flatfish of great commercial value. And there is limited information in the turbot database regarding genes related to the reproductive system. Here, we conducted high-throughput transcriptome profiling of turbot gonad tissues to better understand their reproductive functions and to supply essential gene sequence information for marker-assisted selection programs in the turbot industry. In this study, two gonad libraries representing sex differences in Scophthalmus maximus yielded 453 818 high-quality reads that were assembled into 24 611 contigs and 33 713 singletons by using 454 pyrosequencing, 13 936 contigs and singletons (CS) of which were annotated using BLASTx. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses revealed that various biological functions and processes were associated with many of the annotated CS. Expression analyses showed that 510 genes were differentially expressed in males versus females; 80% of these genes were annotated. In addition, 6484 and 6036 single nucleotide polymorphisms (SNPs) were identified in male and female libraries, respectively. This transcriptome resource will serve as the foundation for cDNA or SNP microarray construction, gene expression characterization, and sex-specific linkage mapping in turbot.

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.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Microarray gene expression profiling using core biopsies of renal neoplasia.

PubMed

Rogers, Craig G; Ditlev, Jonathon A; Tan, Min-Han; Sugimura, Jun; Qian, Chao-Nan; Cooper, Jeff; Lane, Brian; Jewett, Michael A; Kahnoski, Richard J; Kort, Eric J; Teh, Bin T

2009-01-01

We investigate the feasibility of using microarray gene expression profiling technology to analyze core biopsies of renal tumors for classification of tumor histology. Core biopsies were obtained ex-vivo from 7 renal tumors-comprised of four histological subtypes-following radical nephrectomy using 18-gauge biopsy needles. RNA was isolated from these samples and, in the case of biopsy samples, amplified by in vitro transcription. Microarray analysis was then used to quantify the mRNA expression patterns in these samples relative to non-diseased renal tissue mRNA. Genes with significant variation across all non-biopsy tumor samples were identified, and the relationship between tumor and biopsy samples in terms of expression levels of these genes was then quantified in terms of Euclidean distance, and visualized by complete linkage clustering. Final pathologic assessment of kidney tumors demonstrated clear cell renal cell carcinoma (4), oncocytoma (1), angiomyolipoma (1) and adrenalcortical carcinoma (1). Five of the seven biopsy samples were most similar in terms of gene expression to the resected tumors from which they were derived in terms of Euclidean distance. All seven biopsies were assigned to the correct histological class by hierarchical clustering. We demonstrate the feasibility of gene expression profiling of core biopsies of renal tumors to classify tumor histology.

Microarray gene expression profiling using core biopsies of renal neoplasia

PubMed Central

Rogers, Craig G.; Ditlev, Jonathon A.; Tan, Min-Han; Sugimura, Jun; Qian, Chao-Nan; Cooper, Jeff; Lane, Brian; Jewett, Michael A.; Kahnoski, Richard J.; Kort, Eric J.; Teh, Bin T.

2009-01-01

We investigate the feasibility of using microarray gene expression profiling technology to analyze core biopsies of renal tumors for classification of tumor histology. Core biopsies were obtained ex-vivo from 7 renal tumors—comprised of four histological subtypes—following radical nephrectomy using 18-gauge biopsy needles. RNA was isolated from these samples and, in the case of biopsy samples, amplified by in vitro transcription. Microarray analysis was then used to quantify the mRNA expression patterns in these samples relative to non-diseased renal tissue mRNA. Genes with significant variation across all non-biopsy tumor samples were identified, and the relationship between tumor and biopsy samples in terms of expression levels of these genes was then quantified in terms of Euclidean distance, and visualized by complete linkage clustering. Final pathologic assessment of kidney tumors demonstrated clear cell renal cell carcinoma (4), oncocytoma (1), angiomyolipoma (1) and adrenalcortical carcinoma (1). Five of the seven biopsy samples were most similar in terms of gene expression to the resected tumors from which they were derived in terms of Euclidean distance. All seven biopsies were assigned to the correct histological class by hierarchical clustering. We demonstrate the feasibility of gene expression profiling of core biopsies of renal tumors to classify tumor histology. PMID:19966938

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.

Biofunctionalization of surfaces by energetic ion implantation: Review of progress on applications in implantable biomedical devices and antibody microarrays

NASA Astrophysics Data System (ADS)

Bilek, Marcela M. M.

2014-08-01

Despite major research efforts in the field of biomaterials, rejection, severe immune responses, scar tissue and poor integration continue to seriously limit the performance of today's implantable biomedical devices. Implantable biomaterials that interact with their host via an interfacial layer of active biomolecules to direct a desired cellular response to the implant would represent a major and much sought after improvement. Another, perhaps equally revolutionary, development that is on the biomedical horizon is the introduction of cost-effective microarrays for fast, highly multiplexed screening for biomarkers on cell membranes and in a variety of analyte solutions. Both of these advances will rely on effective methods of functionalizing surfaces with bioactive molecules. After a brief introduction to other methods currently available, this review will describe recently developed approaches that use energetic ions extracted from plasma to facilitate simple, one-step covalent surface immobilization of bioactive molecules. A kinetic theory model of the immobilization process by reactions with long-lived, mobile, surface-embedded radicals will be presented. The roles of surface chemistry and microstructure of the ion treated layer will be discussed. Early progress on applications of this technology to create diagnostic microarrays and to engineer bioactive surfaces for implantable biomedical devices will be reviewed.

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

mRNA Expression Profiling of Laser Microbeam Microdissected Cells from Slender Embryonic Structures

PubMed Central

Scheidl, Stefan J.; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

2002-01-01

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-β1, 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. PMID:11891179

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Expression profiling of cardiovascular disease

PubMed Central

2004-01-01

Cardiovascular disease is the most important cause of morbidity and mortality in developed countries, causing twice as many deaths as cancer in the USA. The major cardiovascular diseases, including coronary artery disease (CAD), myocardial infarction (MI), congestive heart failure (CHF) and common congenital heart disease (CHD), are caused by multiple genetic and environmental factors, as well as the interactions between them. The underlying molecular pathogenic mechanisms for these disorders are still largely unknown, but gene expression may play a central role in the development and progression of cardiovascular disease. Microarrays are high-throughput genomic tools that allow the comparison of global expression changes in thousands of genes between normal and diseased cells/tissues. Microarrays have recently been applied to CAD/MI, CHF and CHD to profile changes in gene expression patterns in diseased and non-diseased patients. This same technology has also been used to characterise endothelial cells, vascular smooth muscle cells and inflammatory cells, with or without various treatments that mimic disease processes involved in CAD/MI. These studies have led to the identification of unique subsets of genes associated with specific diseases and disease processes. Ongoing microarray studies in the field will provide insights into the molecular mechanism of cardiovascular disease and may generate new diagnostic and therapeutic markers. PMID:15588496

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.

Global circular RNA expression profile of human gastric cancer and its clinical significance.

PubMed

Shao, Yongfu; Li, Jinyun; Lu, Rongdan; Li, Tianwen; Yang, Yunben; Xiao, Bingxiu; Guo, Junming

2017-06-01

Circular RNAs (circRNAs) are a new class of noncoding RNAs. However, the expression profile and clinical significance of circRNAs in human gastric cancer is unclear. The global circRNA expression profile in human gastric cancer was measured by circRNA microarray. Hsa_circ_0014717, one of the most downregulated circRNAs in microarray, was selected as a targeted circRNA to explore its levels in gastric tissues and gastric juice. Freeze-thaw experiment and incubation experiment confirmed the stability of gastric juice circRNAs. A total of 308 circRNAs, including 107 (34.74%) upregulated and 201 (65.26%) downregulated circRNAs, were found significantly aberrantly expressed in gastric cancer tissues. The top ten upregulated in gastric cancer tissues were hsa_circ_0035445, hsa_circ_0003789, hsa_circ_0063809, hsa_circ_0074362, hsa_circ_0006282, hsa_circ_0011107, hsa_circ_0084606, hsa_circ_0005556, hsa_circ_0050547, and hsa_circ_0006470, while the top ten downregulated ones were hsa_circ_0007099, hsa_circ_0001897, hsa_circ_0007707, hsa_circ_0008832, hsa_circ_0001546, hsa_circ_0002089, hsa_circ_0004680, hsa_circ_0000154, hsa_circ_0004458, and hsa_circ_0008394. The hot-point chromosomes were chr1, chr2, chr3, chr9, and chr17. Hsa_circ_0014717 was significantly downregulated in 77.2% (74/96) gastric cancer tissues. Its levels in gastric cancer tissues were related to tumor stage (P = 0.037), distal metastasis (P = 0.048), tissue carcinoembryonic antigen (P = 0.001), and carbohydrate antigen 19-9 expression (P = 0.021). More importantly, hsa_circ_0014717 can stably exist in human gastric juice; and its nature meets the requirements of clinical detection. Our study uncovered the circRNA expression profile in human gastric cancer. Moreover, some circRNAs can stably exist in human body fluid, and has the potential to be used as novel biomarkers for the screening of high-risk gastric cancer patients. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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.

Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers.

PubMed

Wicik, Z; Gajewska, M; Majewska, A; Walkiewicz, D; Osińska, E; Motyl, T

2016-02-01

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR-101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF-beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern. © 2015 Blackwell Verlag GmbH.

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

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.

Troyer Syndrome

MedlinePlus

... Coordinating Committees CounterACT Rigor & Transparency Scientific Resources Animal Models Cell/Tissue/DNA Clinical and Translational Resources Gene ... Coordinating Committees CounterACT Rigor & Transparency Scientific Resources Animal Models Cell/Tissue/DNA Clinical and Translational Resources Gene ...

Transient Ischemic Attack

MedlinePlus

... Coordinating Committees CounterACT Rigor & Transparency Scientific Resources Animal Models Cell/Tissue/DNA Clinical and Translational Resources Gene ... Coordinating Committees CounterACT Rigor & Transparency Scientific Resources Animal Models Cell/Tissue/DNA Clinical and Translational Resources Gene ...

Downregulation of P-cadherin expression in hepatocellular carcinoma induces tumorigenicity

PubMed Central

Bauer, Richard; Valletta, Daniela; Bauer, Karin; Thasler, Wolfgang E; Hartmann, Arndt; Müller, Martina; Reichert, Torsten E; Hellerbrand, Claus

2014-01-01

P-cadherin is a major contributor to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes and in maintaining tissue integrity and homeostasis. Alterations of P-cadherin expression have been observed during the progression of several carcinomas where it appears to act as tumor suppressive or oncogenic in a context-dependent manner. Here, we found a significant downregulation of P-cadherin in hepatocellular carcinoma (HCC) cell lines and tissues compared to primary human hepatocytes and non-malignant liver tissues. Combined immunohistochemical analysis of a tissue microarray containing matched pairs of HCC tissue and corresponding non-tumorous liver tissue of 69 patients confirmed reduced P-cadherin expression in more than half of the cases. In 35 human HCC tissues, the P-cadherin immunosignal was completely lost which correlated with tumor staging and proliferation. Also in vitro, P-cadherin suppression in HCC cells via siRNA induced proliferation compared to cells transfected with control-siRNA. In summary, downregulation of P-cadherin expression appears to induce tumorigenicity in HCC. Therefore, P-cadherin expression may serve as a prognostic marker and therapeutic target of this highly aggressive tumor. PMID:25337260

Downregulation of P-cadherin expression in hepatocellular carcinoma induces tumorigenicity.

PubMed

Bauer, Richard; Valletta, Daniela; Bauer, Karin; Thasler, Wolfgang E; Hartmann, Arndt; Müller, Martina; Reichert, Torsten E; Hellerbrand, Claus

2014-01-01

P-cadherin is a major contributor to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes and in maintaining tissue integrity and homeostasis. Alterations of P-cadherin expression have been observed during the progression of several carcinomas where it appears to act as tumor suppressive or oncogenic in a context-dependent manner. Here, we found a significant downregulation of P-cadherin in hepatocellular carcinoma (HCC) cell lines and tissues compared to primary human hepatocytes and non-malignant liver tissues. Combined immunohistochemical analysis of a tissue microarray containing matched pairs of HCC tissue and corresponding non-tumorous liver tissue of 69 patients confirmed reduced P-cadherin expression in more than half of the cases. In 35 human HCC tissues, the P-cadherin immunosignal was completely lost which correlated with tumor staging and proliferation. Also in vitro, P-cadherin suppression in HCC cells via siRNA induced proliferation compared to cells transfected with control-siRNA. In summary, downregulation of P-cadherin expression appears to induce tumorigenicity in HCC. Therefore, P-cadherin expression may serve as a prognostic marker and therapeutic target of this highly aggressive tumor.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

PubMed

Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

2016-08-21

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Spectral Analysis of Breast Cancer on Tissue Microarrays: Seeing Beyond Morphology

DTIC Science & Technology

2005-04-01

Harvey N., Szymanski J.J., Bloch J.J., Mitchell M. investigation of image feature extraction by a genetic algorithm. Proc. SPIE 1999;3812:24-31. 11...automated feature extraction using multiple data sources. Proc. SPIE 2003;5099:190-200. 15 4 Spectral-Spatial Analysis of Urine Cytology Angeletti et al...Appendix Contents: 1. Harvey, N.R., Levenson, R.M., Rimm, D.L. (2003) Investigation of Automated Feature Extraction Techniques for Applications in

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

Farahani, Poupak; Chiu, Sally; Bowlus, Christopher L.

Obesity is a complex disease. To date, over 100 chromosomal loci for body weight, body fat, regional white adipose tissue weight, and other obesity-related traits have been identified in humans and in animal models. For most loci, the underlying genes are not yet identified; some of these chromosomal loci will be alleles of known obesity genes, whereas many will represent alleles of unknown genes. Microarray analysis allows simultaneous multiple gene and pathway discovery. cDNA and oligonucleotide arrays are commonly used to identify differentially expressed genes by surveys of large numbers of known and unnamed genes. Two papers previously identified genesmore » differentially expressed in adipose tissue of mouse models of obesity and diabetes by analysis of hybridization to Affymetrix oligonucleotide chips.« less

Malignant lymphoma in a West Indian manatee (Trichechus manatus).

PubMed

Hammer, Anne S; Klausen, Bjarne; Knold, Steffen; Dietz, Hans H; Dutoit, Stephen J Hamilton

2005-10-01

We identified a malignant lymphoma infiltrating the lung, liver, kidney, mesenteric lymph nodes, and eye as the cause of death in a male West Indian manatee (Trichechus manatus). Diagnosis was based on gross, histopathologic, and immunohistochemical studies. Tissue samples from ten organs were included in a tissue microarray and sections from this array were subjected to immunohistochemical staining. The cytoplasm of the neoplastic lymphocytes identified in six organs was positive for CD3, a marker for T-cell differentiation. The neoplastic cells were negative for CD79alpha, a marker for B-cell differentiation. The cause of this neoplasm was not determined. This is the first report of malignant lymphoma in the mammal order Sirenia.

Assessing Global Transcriptome Changes in Response to South African Cassava Mosaic Virus [ZA-99] Infection in Susceptible Arabidopsis thaliana.

PubMed

Pierce, Erica J; Rey, M E Chrissie

2013-01-01

In susceptible plant hosts, co-evolution has favoured viral strategies to evade host defenses and utilize resources to their own benefit. The degree of manipulation of host gene expression is dependent on host-virus specificity and certain abiotic factors. In order to gain insight into global transcriptome changes for a geminivirus pathosystem, South African cassava mosaic virus [ZA:99] and Arabidopsis thaliana, 4×44K Agilent microarrays were adopted. After normalization, a log2 fold change filtering of data (p<0.05) identified 1,743 differentially expressed genes in apical leaf tissue. A significant increase in differential gene expression over time correlated with an increase in SACMV accumulation, as virus copies were 5-fold higher at 24 dpi and 6-fold higher at 36 dpi than at 14 dpi. Many altered transcripts were primarily involved in stress and defense responses, phytohormone signalling pathways, cellular transport, cell-cycle regulation, transcription, oxidation-reduction, and other metabolic processes. Only forty-one genes (2.3%) were shown to be continuously expressed across the infection period, indicating that the majority of genes were transient and unique to a particular time point during infection. A significant number of pathogen-responsive genes were suppressed during the late stages of pathogenesis, while during active systemic infection (14 to 24 dpi), there was an increase in up-regulated genes in several GO functional categories. An adaptive response was initiated to divert energy from growth-related processes to defense, leading to disruption of normal biological host processes. Similarities in cell-cycle regulation correlated between SACMV and Cabbage leaf curl virus (CaLCuV), but differences were also evident. Differences in gene expression between the two geminiviruses clearly demonstrated that, while some global transcriptome responses are generally common in plant virus infections, temporal host-specific interactions are required for successful geminivirus infection. To our knowledge this is the first geminivirus microarray study identifying global differentially expressed transcripts at 3 time points.

Assessing Global Transcriptome Changes in Response to South African Cassava Mosaic Virus [ZA-99] Infection in Susceptible Arabidopsis thaliana

PubMed Central

Pierce, Erica J.; Rey, M. E. Chrissie

2013-01-01

In susceptible plant hosts, co-evolution has favoured viral strategies to evade host defenses and utilize resources to their own benefit. The degree of manipulation of host gene expression is dependent on host-virus specificity and certain abiotic factors. In order to gain insight into global transcriptome changes for a geminivirus pathosystem, South African cassava mosaic virus [ZA:99] and Arabidopsis thaliana, 4×44K Agilent microarrays were adopted. After normalization, a log2 fold change filtering of data (p<0.05) identified 1,743 differentially expressed genes in apical leaf tissue. A significant increase in differential gene expression over time correlated with an increase in SACMV accumulation, as virus copies were 5-fold higher at 24 dpi and 6-fold higher at 36 dpi than at 14 dpi. Many altered transcripts were primarily involved in stress and defense responses, phytohormone signalling pathways, cellular transport, cell-cycle regulation, transcription, oxidation-reduction, and other metabolic processes. Only forty-one genes (2.3%) were shown to be continuously expressed across the infection period, indicating that the majority of genes were transient and unique to a particular time point during infection. A significant number of pathogen-responsive genes were suppressed during the late stages of pathogenesis, while during active systemic infection (14 to 24 dpi), there was an increase in up-regulated genes in several GO functional categories. An adaptive response was initiated to divert energy from growth-related processes to defense, leading to disruption of normal biological host processes. Similarities in cell-cycle regulation correlated between SACMV and Cabbage leaf curl virus (CaLCuV), but differences were also evident. Differences in gene expression between the two geminiviruses clearly demonstrated that, while some global transcriptome responses are generally common in plant virus infections, temporal host-specific interactions are required for successful geminivirus infection. To our knowledge this is the first geminivirus microarray study identifying global differentially expressed transcripts at 3 time points. PMID:23826319

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

Stanniocalcin 2 is an estrogen-responsive gene coexpressed with the estrogen receptor in human breast cancer.

PubMed

Bouras, Toula; Southey, Melissa C; Chang, Andy C; Reddel, Roger R; Willhite, Dorian; Glynne, Richard; Henderson, Michael A; Armes, Jane E; Venter, Deon J

2002-03-01

Differences in gene expression are likely to explain the phenotypic variation between hormone-responsive and hormone-unresponsive breast cancers. In this study, DNA microarray analysis of approximately 10,000 known genes and 25,000 expressed sequence tag clusters was performed to identify genes induced by estrogen and repressed by the pure antiestrogen ICI 182 780 in vitro that correlated with estrogen receptor (ER) expression in primary breast carcinomas in vivo. Stanniocalcin (STC) 2 was identified as one of the genes that fulfilled these criteria. DNA microarray hybridization showed a 3-fold induction of STC2 mRNA expression in MCF-7 cells in < or = 3 h of estrogen exposure and a 3-fold repression in the presence of antiestrogen (one-way ANOVA, P < 0.0005). In 13 ER-positive and 12 ER-negative breast carcinomas, the microarray-derived mRNA levels observed for STC2 correlated with tumor ER mRNA (Pearson's correlation, r = 0.85; P < 0.0001) and ER protein status (Spearman's rank correlation, r = 0.73; P < 0.0001). The expression profile of STC2 was further confirmed by in situ hybridization and immunohistochemistry on a larger cohort of 236 unselected breast carcinomas using tissue microarrays. STC2 mRNA and protein expression were found to be associated with tumor ER status (Fisher's exact test, P < 0.005). The related gene, STC1, was also examined and shown to be associated with ER status in breast carcinomas (Fisher's exact test, P < 0.05). This study demonstrates the feasibility of using global gene expression data derived from an in vitro model to pinpoint novel estrogen-responsive genes of potential clinical relevance.

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.

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

Samolski, Ilanit; de Luis, Alberto; Vizcaíno, Juan Antonio; Monte, Enrique; Suárez, M Belén

2009-10-13

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. 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. 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.

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

Analysis of baseline gene expression levels from ...

EPA Pesticide Factsheets

The use of gene expression profiling to predict chemical mode of action would be enhanced by better characterization of variance due to individual, environmental, and technical factors. Meta-analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies has yielded useful information on baseline fluctuations in gene expression. A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques. The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selectiv

Development and Application of a Salmonid EST Database and cDNA Microarray: Data Mining and Interspecific Hybridization Characteristics

PubMed Central

Rise, Matthew L.; von Schalburg, Kristian R.; Brown, Gordon D.; Mawer, Melanie A.; Devlin, Robert H.; Kuipers, Nathanael; Busby, Maura; Beetz-Sargent, Marianne; Alberto, Roberto; Gibbs, A. Ross; Hunt, Peter; Shukin, Robert; Zeznik, Jeffrey A.; Nelson, Colleen; Jones, Simon R.M.; Smailus, Duane E.; Jones, Steven J.M.; Schein, Jacqueline E.; Marra, Marco A.; Butterfield, Yaron S.N.; Stott, Jeff M.; Ng, Siemon H.S.; Davidson, William S.; Koop, Ben F.

2004-01-01

We report 80,388 ESTs from 23 Atlantic salmon (Salmo salar) cDNA libraries (61,819 ESTs), 6 rainbow trout (Oncorhynchus mykiss) cDNA libraries (14,544 ESTs), 2 chinook salmon (Oncorhynchus tshawytscha) cDNA libraries (1317 ESTs), 2 sockeye salmon (Oncorhynchus nerka) cDNA libraries (1243 ESTs), and 2 lake whitefish (Coregonus clupeaformis) cDNA libraries (1465 ESTs). The majority of these are 3′ sequences, allowing discrimination between paralogs arising from a recent genome duplication in the salmonid lineage. Sequence assembly reveals 28,710 different S. salar, 8981 O. mykiss, 1085 O. tshawytscha, 520 O. nerka, and 1176 C. clupeaformis putative transcripts. We annotate the submitted portion of our EST database by molecular function. Higher- and lower-molecular-weight fractions of libraries are shown to contain distinct gene sets, and higher rates of gene discovery are associated with higher-molecular weight libraries. Pyloric caecum library group annotations indicate this organ may function in redox control and as a barrier against systemic uptake of xenobiotics. A microarray is described, containing 7356 salmonid elements representing 3557 different cDNAs. Analyses of cross-species hybridizations to this cDNA microarray indicate that this resource may be used for studies involving all salmonids. PMID:14962987

DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

PubMed Central

Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

2015-01-01

Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

Using glycome databases for drug discovery.

PubMed

Aoki-Kinoshita, Kiyoko F

2008-08-01

The glycomics field has made great advancements in the last decade due to technologies for their synthesis and analysis including carbohydrate microarrays. Accordingly, databases for glycomics research have also emerged and been made publicly available by many major institutions worldwide. This review introduces these and other useful databases on which new methods for drug discovery can be developed. The scope of this review covers current documented and accessible databases and resources pertaining to glycomics. These were selected with the expectation that they may be useful for drug discovery research. There is a plethora of glycomics databases that have much potential for drug discovery. This may seem daunting at first but this review helps to put some of these resources into perspective. Additionally, some thoughts on how to integrate these resources to allow more efficient research are presented.

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

The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate Cancer Treatment Assignment

DTIC Science & Technology

2017-10-01

PI Mohler). Immunostaining of prostate cancer biopsy specimen of patients who qualified for active surveillance is complete (PI Goodrich). ELISA assay...specimens for use in constructing tissue microarrays, developing and optimizing ELISA assays to detect Thoc1 protein and anti-Thoc1 autoantibodies...set of TMAs made from 700 patients available at RPCI has been immunostained for PMP22 (figure 5). ELISA assays for measuring pThoc1 and pThoc1

The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate Cancer Treatment Assignment

DTIC Science & Technology

2016-10-01

patients on active surveillance is ongoing (PI Mohler). Developing ELISA assays for measuring pThoc1 and pThoc1 autoantibodies is complete (PI...assessment of 1146 patient specimens for use in constructing tissue microarrays, developing and optimizing ELISA assays to detect anti-Thoc1 autoantibodies...700 patients available at RPCI has been immunostained for PMP22 (figure 3). ELISA assays for measuring pThoc1 and pThoc1 autoantibodies have been