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Sample records for progression tissue microarray

  1. Tissue Microarrays.

    PubMed

    Dancau, Ana-Maria; Simon, Ronald; Mirlacher, Martina; Sauter, Guido

    2016-01-01

    Modern next-generation sequencing and microarray technologies allow for the simultaneous analysis of all human genes on the DNA, RNA, miRNA, and methylation RNA level. Studies using such techniques have lead to the identification of hundreds of genes with a potential role in cancer or other diseases. The validation of all of these candidate genes requires in situ analysis of high numbers of clinical tissues samples. The tissue microarray technology greatly facilitates such analysis. In this method minute tissue samples (typically 0.6 mm in diameter) from up to 1000 different tissues can be analyzed on one microscope glass slide. All in situ methods suitable for histological studies can be applied to TMAs without major changes of protocols, including immunohistochemistry, fluorescence in situ hybridization, or RNA in situ hybridization. Because all tissues are analyzed simultaneously with the same batch of reagents, TMA studies provide an unprecedented degree of standardization, speed, and cost efficiency.

  2. A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers.

    PubMed

    Camp, Robert L; Neumeister, Veronique; Rimm, David L

    2008-12-01

    This year, 2008, marks the 10-year anniversary of the development of the modern tissue microarray (TMA). During the last decade, the use of TMAs has grown steadily and accounts for a small but increasing percentage of all cancer biomarker studies performed. The growing popularity of TMA-based studies attests to their benefits in the discovery and validation of new biomarkers. This review will focus on these benefits, but also on the faults of TMAs and the challenges of TMA studies that have been overcome in the last decade. We will also discuss the role of TMAs in the latest revolution in cancer treatment, the use of targeted drug therapy.

  3. Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma

    PubMed Central

    Xue, Li-yan; Hu, Nan; Song, Yong-mei; Zou, Shuang-mei; Shou, Jian-zhong; Qian, Lu-xia; Ren, Li-qun; Lin, Dong-mei; Tong, Tong; He, Zu-gen; Zhan, Qi-min; Taylor, Philip R; Lu, Ning

    2006-01-01

    Background The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC. PMID:17187659

  4. Tissue Microarrays in Clinical Oncology

    PubMed Central

    Voduc, David; Kenney, Challayne; Nielsen, Torsten O.

    2008-01-01

    The tissue microarray is a recently-implemented, high-throughput technology for the analysis of molecular markers in oncology. This research tool permits the rapid assessment of a biomarker in thousands of tumor samples, using commonly available laboratory assays such as immunohistochemistry and in-situ hybridization. Although introduced less than a decade ago, the TMA has proven to be invaluable in the study of tumor biology, the development of diagnostic tests, and the investigation of oncological biomarkers. This review describes the impact of TMA-based research in clinical oncology and its potential future applications. Technical aspects of TMA construction, and the advantages and disadvantages inherent to this technology are also discussed. PMID:18314063

  5. Segmentation of prostate cancer tissue microarray images

    NASA Astrophysics Data System (ADS)

    Cline, Harvey E.; Can, Ali; Padfield, Dirk

    2006-02-01

    Prostate cancer is diagnosed by histopathology interpretation of hematoxylin and eosin (H and E)-stained tissue sections. Gland and nuclei distributions vary with the disease grade. The morphological features vary with the advance of cancer where the epithelial regions grow into the stroma. An efficient pathology slide image analysis method involved using a tissue microarray with known disease stages. Digital 24-bit RGB images were acquired for each tissue element on the slide with both 10X and 40X objectives. Initial segmentation at low magnification was accomplished using prior spectral characteristics from a training tissue set composed of four tissue clusters; namely, glands, epithelia, stroma and nuclei. The segmentation method was automated by using the training RGB values as an initial guess and iterating the averaging process 10 times to find the four cluster centers. Labels were assigned to the nearest cluster center in red-blue spectral feature space. An automatic threshold algorithm separated the glands from the tissue. A visual pseudo color representation of 60 segmented tissue microarray image was generated where white, pink, red, blue colors represent glands, epithelia, stroma and nuclei, respectively. The higher magnification images provided refined nuclei morphology. The nuclei were detected with a RGB color space principle component analysis that resulted in a grey scale image. The shape metrics such as compactness, elongation, minimum and maximum diameters were calculated based on the eigenvalues of the best-fitting ellipses to the nuclei.

  6. Tissue microarray profiling in human heart failure.

    PubMed

    Lal, Sean; Nguyen, Lisa; Tezone, Rhenan; Ponten, Fredrik; Odeberg, Jacob; Li, Amy; Dos Remedios, Cristobal

    2016-09-01

    Tissue MicroArrays (TMAs) are a versatile tool for high-throughput protein screening, allowing qualitative analysis of a large number of samples on a single slide. We have developed a customizable TMA system that uniquely utilizes cryopreserved human cardiac samples from both heart failure and donor patients to produce formalin-fixed paraffin-embedded sections. Confirmatory upstream or downstream molecular studies can then be performed on the same (biobanked) cryopreserved tissue. In a pilot study, we applied our TMAs to screen for the expression of four-and-a-half LIM-domain 2 (FHL2), a member of the four-and-a-half LIM family. This protein has been implicated in the pathogenesis of heart failure in a variety of animal models. While FHL2 is abundant in the heart, not much is known about its expression in human heart failure. For this purpose, we generated an affinity-purified rabbit polyclonal anti-human FHL2 antibody. Our TMAs allowed high-throughput profiling of FHL2 protein using qualitative and semiquantitative immunohistochemistry that proved complementary to Western blot analysis. We demonstrated a significant relative reduction in FHL2 protein expression across different forms of human heart failure.

  7. Progress in the application of DNA microarrays.

    PubMed Central

    Lobenhofer, E K; Bushel, P R; Afshari, C A; Hamadeh, H K

    2001-01-01

    Microarray technology has been applied to a variety of different fields to address fundamental research questions. The use of microarrays, or DNA chips, to study the gene expression profiles of biologic samples began in 1995. Since that time, the fundamental concepts behind the chip, the technology required for making and using these chips, and the multitude of statistical tools for analyzing the data have been extensively reviewed. For this reason, the focus of this review will be not on the technology itself but on the application of microarrays as a research tool and the future challenges of the field. PMID:11673116

  8. [A novel automatic manufacture device for tissue micro-array].

    PubMed

    Wang, Chaohui; Chen, Chao; Zhang, Qunming; Jiang, Zhuangde; Wang, Teng; Meng, Tao

    2007-10-01

    A novel automatic manufacture device for tissue micro-array is introduced in this paper. Based on the analyses of task and process, the new device prototype is researched and developed. The device consists of a paraffin positioning module and a three-manipulator module. The control system is composed of accurate navigation sub-system, digital image recognition sub-system and punching-filling operating sub-system. The results of experiment demonstrate that the device can accomplish the operations such as image automatic recognition, accurate position, auto-punching and filling. It fulfills the requirements to automatic manufacture of tissue micro-array. PMID:18027675

  9. Examination of Oral Cancer Biomarkers by Tissue Microarray Analysis

    PubMed Central

    Choi, Peter; Jordan, C. Diana; Mendez, Eduardo; Houck, John; Yueh, Bevan; Farwell, D. Gregory; Futran, Neal; Chen, Chu

    2008-01-01

    Background Oral squamous cell carcinoma (OSCC) is a major healthcare problem worldwide. Efforts in our laboratory and others focusing on the molecular characterization of OSCC tumors with the use of DNA microarrays have yielded heterogeneous results. To validate the DNA microarray results on a subset of genes from these studies that could potentially serve as biomarkers of OSCC, we elected to examine their expression by an alternate quantitative method and by assessing their protein levels. Design Based on DNA microarray data from our lab and data reported in the literature, we identified six potential biomarkers of OSCC to investigate further. We employed quantitative, real-time polymerase chain reaction (qRT-PCR) to examine expression changes of CDH11, MMP3, SPARC, POSTN, TNC, TGM3 in OSCC and normal control tissues. We further examined validated markers on the protein level by immunohistochemistry (IHC) analysis of OSCC tissue microarray (TMA) sections. Results qRT-PCR analysis revealed up-regulation of CDH11, SPARC, POSTN, and TNC gene expression, and decreased TGM3 expression in OSCC compared to normal controls. MMP3 was not found to be differentially expressed. In TMA IHC analyses, SPARC, periostin, and tenascin C exhibited increased protein expression in cancer compared to normal tissues, and their expression was primarily localized within tumor-associated stroma rather than tumor epithelium. Conversely, transglutaminase-3 protein expression was found only within keratinocytes in normal controls, and was significantly down-regulated in cancer cells. Conclusions Of six potential gene markers of OSCC, initially identified by DNA microarray analyses, differential expression of CDH11, SPARC, POSTN, TNC, and TGM3 were validated by qRT-PCR. Differential expression and localization of proteins encoded by SPARC, POSTN, TNC, and TGM3 were clearly shown by TMA IHC. PMID:18490578

  10. Producing reverse phase protein microarrays from formalin-fixed tissues.

    PubMed

    Wolff, Claudia; Schott, Christina; Malinowsky, Katharina; Berg, Daniela; Becker, Karl-Friedrich

    2011-01-01

    In most hospitals around the world FFPE (formalin fixed, paraffin embedded) tissues have been used for diagnosis and have subsequently been archived since decades. This has lead to a sizeable pool of this kind of tissues. Till quite recently it was not possible to use this congeries of samples for protein analysis, but now several groups described successful protein extraction from FFPE tissues. In this chapter, we describe a protein extraction protocol established in our laboratory combined with the use of reverse phase protein microarray.

  11. Application of Tissue Microarray Technology to Stem Cell Research

    PubMed Central

    Spada, Alberto La; Rainoldi, Barnaba; Blasio, Andrea De; Biunno, Ida

    2014-01-01

    There is virtually an unlimited number of possible Tissue Microarray (TMA) applications in basic and clinical research and ultimately in diagnostics. However, to assess the functional importance of novel markers, researchers very often turn to cell line model systems. The appropriate choice of a cell line is often a difficult task, but the use of cell microarray (CMA) technology enables a quick screening of several markers in cells of different origins, mimicking a genomic-scale analysis. In order to improve the morphological evaluations of the CMA slides we harvested the cells by conventional trypsinization, mechanical scraping and cells grown on coverslips. We show that mechanical scraping is a good evaluation method since keeps the real morphology very similar to those grown on coverslips. Immunofluorescence images are of higher quality, facilitating the reading of the biomarker cellular and subcellular localization. Here, we describe CMA technology in stem cell research.

  12. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  13. Automated prostate cancer diagnosis and Gleason grading of tissue microarrays

    NASA Astrophysics Data System (ADS)

    Tabesh, Ali; Kumar, Vinay P.; Pang, Ho-Yuen; Verbel, David; Kotsianti, Angeliki; Teverovskiy, Mikhail; Saidi, Olivier

    2005-04-01

    We present the results on the development of an automated system for prostate cancer diagnosis and Gleason grading. Images of representative areas of the original Hematoxylin-and-Eosin (H&E)-stained tissue retrieved from each patient, either from a tissue microarray (TMA) core or whole section, were captured and analyzed. The image sets consisted of 367 and 268 color images for the diagnosis and Gleason grading problems, respectively. In diagnosis, the goal is to classify a tissue image into tumor versus non-tumor classes. In Gleason grading, which characterizes tumor aggressiveness, the objective is to classify a tissue image as being from either a low- or high-grade tumor. Several feature sets were computed from the image. The feature sets considered were: (i) color channel histograms, (ii) fractal dimension features, (iii) fractal code features, (iv) wavelet features, and (v) color, shape and texture features computed using Aureon Biosciences' MAGIC system. The linear and quadratic Gaussian classifiers together with a greedy search feature selection algorithm were used. For cancer diagnosis, a classification accuracy of 94.5% was obtained on an independent test set. For Gleason grading, the achieved accuracy of classification into low- and high-grade classes of an independent test set was 77.6%.

  14. Classification and immunohistochemical scoring of breast tissue microarray spots.

    PubMed

    Amaral, Telmo; McKenna, Stephen J; Robertson, Katherine; Thompson, Alastair

    2013-10-01

    Tissue microarrays (TMAs) facilitate the survey of very large numbers of tumors. However, the manual assessment of stained TMA sections constitutes a bottleneck in the pathologist's work flow. This paper presents a computational pipeline for automatically classifying and scoring breast cancer TMA spots that have been subjected to nuclear immunostaining. Spots are classified based on a bag of visual words approach. Immunohistochemical scoring is performed by computing spot features reflecting the proportion of epithelial nuclei that are stained and the strength of that staining. These are then mapped onto an ordinal scale used by pathologists. Multilayer perceptron classifiers are compared with latent topic models and support vector machines for spot classification, and with Gaussian process ordinal regression and linear models for scoring. Intraobserver variation is also reported. The use of posterior entropy to identify uncertain cases is demonstrated. Evaluation is performed using TMA images stained for progesterone receptor.

  15. TAMEE: data management and analysis for tissue microarrays

    PubMed Central

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

    2007-01-01

    Background 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. Results 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. Conclusion 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 . PMID:17343750

  16. An adapted tissue microarray for the development of a matrix arrangement of tissue samples.

    PubMed

    Gurgel, Daniel C; Dornelas, Conceição A; Lima-Júnior, Roberto C P; Ribeiro, Ronaldo A; Almeida, Paulo R C

    2012-03-15

    The arrangement of tissue samples in a matrix, known as the tissue microarray (TMA) method, is a well-recognized method worldwide. This technique makes it possible to assess the expression of molecular markers on a large scale with high yields in terms of time, costs, and archived material. Some researchers are trying to adapt the technique to expand the research possibilities. This study proposes an adaptive simplification of low-cost instruments for obtaining samples that will be used in the construction of the TMA. The use of a manual leather puncher, which has a very low cost and a long expected life and eliminates the need to use a press machine, is a simple and effective alternative to building blocks of tissue microarrays.

  17. 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

  18. Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.

    PubMed

    Henke, R T; Maitra, A; Paik, S; Wellstein, A

    2005-01-01

    Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.

  19. TMAinspiration: Decode Interdependencies in Multifactorial Tissue Microarray Data.

    PubMed

    Boecker, Florian; Buerger, Horst; Mallela, Nikhil V; Korsching, Eberhard

    2016-01-01

    There are no satisfying tools in tissue microarray (TMA) data analysis up to now to analyze the cooperative behavior of all measured markers in a multifactorial TMA approach. The developed tool TMAinspiration is not only offering an analysis option to close this gap but also offering an ecosystem consisting of quality control concepts and supporting scripts to make this approach a platform for informed practice and further research. The TMAinspiration method is specifically focusing on the demands of the TMA analysis by controlling errors and noise by a generalized regression scheme while at the same time avoiding to introduce a priori too many constraints into the analysis of the data. So, we are testing partitions of a proximity table to find an optimal support for a ranking scheme of molecular dependencies. The idea of combining several partitions to one ensemble, which is balancing the optimization process, is based on the main assumption that all these perspectives on the cellular network need to be self-consistent. Several application examples in breast cancer and one in squamous cell carcinoma demonstrate that this procedure is nicely confirming a priori knowledge on the expression characteristics of protein markers, while also integrating many new results discovered in the treasury of a bigger TMA experiment. The code and software are now freely available at: http://complex-systems.uni-muenster.de/tma_inspiration.html. PMID:27398021

  20. TMAinspiration: Decode Interdependencies in Multifactorial Tissue Microarray Data

    PubMed Central

    Boecker, Florian; Buerger, Horst; Mallela, Nikhil V.; Korsching, Eberhard

    2016-01-01

    There are no satisfying tools in tissue microarray (TMA) data analysis up to now to analyze the cooperative behavior of all measured markers in a multifactorial TMA approach. The developed tool TMAinspiration is not only offering an analysis option to close this gap but also offering an ecosystem consisting of quality control concepts and supporting scripts to make this approach a platform for informed practice and further research. The TMAinspiration method is specifically focusing on the demands of the TMA analysis by controlling errors and noise by a generalized regression scheme while at the same time avoiding to introduce a priori too many constraints into the analysis of the data. So, we are testing partitions of a proximity table to find an optimal support for a ranking scheme of molecular dependencies. The idea of combining several partitions to one ensemble, which is balancing the optimization process, is based on the main assumption that all these perspectives on the cellular network need to be self-consistent. Several application examples in breast cancer and one in squamous cell carcinoma demonstrate that this procedure is nicely confirming a priori knowledge on the expression characteristics of protein markers, while also integrating many new results discovered in the treasury of a bigger TMA experiment. The code and software are now freely available at: http://complex-systems.uni-muenster.de/tma_inspiration.html. PMID:27398021

  1. Fluorescence in situ hybridization analysis of formalin fixed paraffin embedded tissues, including tissue microarrays.

    PubMed

    Summersgill, Brenda M; Shipley, Janet M

    2010-01-01

    Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2-3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources.

  2. Identification of different subtypes of breast cancer using tissue microarray.

    PubMed

    Munirah, M A; Siti-Aishah, M A; Reena, M Z; Sharifah, N A; Rohaizak, M; Norlia, A; Rafie, M K M; Asmiati, A; Hisham, A; Fuad, I; Shahrun, N S; Das, S

    2011-01-01

    Breast cancer may be classified into luminal A, luminal B, HER2+/ER-, basal-like and normal-like subtypes based on gene expression profiling or immunohistochemical (IHC) characteristics. The main aim of the present study was to classify breast cancer into molecular subtypes based on immunohistochemistry findings and correlate the subtypes with clinicopathological factors. Two hundred and seventeen primary breast carcinomas tumor tissues were immunostained for ER, PR, HER2, CK5/6, EGFR, CK8/18, p53 and Ki67 using tissue microarray technique. All subtypes were significantly associated with Malay ethnic background (p=0.035) compared to other racial origins. The most common subtypes of breast cancers were luminal A and was significantly associated with low histological grade (p<0.000) and p53 negativity (p=0.003) compared to HER2+/ER-, basal-like and normal-like subtypes with high histological grade (p<0.000) and p53 positivity (p=0.003). Luminal B subtype had the smallest mean tumor size (p=0.009) and also the highest mean number of lymph nodes positive (p=0.032) compared to other subtypes. All markers except EGFR and Ki67 were significantly associated with the subtypes. The most common histological type was infiltrating ductal carcinoma, NOS. Majority of basal-like subtype showed comedo-type necrosis (68.8%) and infiltrative margin (81.3%). Our studies suggest that IHC can be used to identify the different subtypes of breast cancer and all subtypes were significantly associated with race, mean tumor size, mean number of lymph node positive, histological grade and all immunohistochemical markers except EGFR and Ki67.

  3. 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.

  4. [Research progress of probe design software of oligonucleotide microarrays].

    PubMed

    Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

    2014-02-01

    DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software.

  5. Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

    PubMed Central

    Ambepitiya Wickramasinghe, Iresha N.; de Vries, Robert P.; Eggert, Amber M.; Wandee, Nantaporn; de Haan, Cornelis A. M.; Gröne, Andrea; Verheije, Monique H.

    2015-01-01

    The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens. PMID:26035584

  6. Cell-based microarrays: current progress, future prospects.

    PubMed

    Palmer, Ella; Freeman, Tom

    2005-07-01

    Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a novel method for performing high-throughput screens of gene function. In this study, expression vectors containing the open reading frame of human genes were printed onto glass microscope slides to form a microarray. Transfection reagents were added pre- or post-spotting, and cells grown over the surface of the array. They demonstrated that cells growing in the immediate vicinity of the expression vectors underwent 'reverse transfection', and that subsequent alterations in cell function could then be detected by secondary assays performed on the array. Subsequent publications have adapted the technique to a variety of applications, and have also shown that the approach works when arrays are fabricated using short interfering RNAs and compounds. The potential of this method for performing analyses of gene function and for identifying novel therapeutic agents has been clearly demonstrated, and current efforts are focused on improving and harnessing this technology for high-throughput screening applications. PMID:16014002

  7. 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.

  8. Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts.

    PubMed

    Catchpoole, D; Mackie, N; McIver, S; Chetcuti, A; Henwood, A; Graf, N; Arbuckle, S

    2011-12-01

    Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape "window" over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.

  9. 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.

  10. Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues.

    PubMed

    Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal

    2016-03-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples. PMID:26981433

  11. Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues.

    PubMed

    Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal

    2016-03-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples.

  12. Phosphoprotein Stability in Clinical Tissue and Its Relevance for Reverse Phase Protein Microarray Technology

    PubMed Central

    Espina, Virginia; Mueller, Claudius; Liotta, Lance A.

    2013-01-01

    Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor’s growth. Reverse Phase Protein Microarrays (RPMA) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and preservation may cause significant variability and bias in downstream molecular analysis. Depending on the ex vivo delay time in tissue processing, and the manner of tissue handling, protein biomarkers such as signal pathway phosphoproteins will be elevated or suppressed in a manner that does not represent the biomarker levels at the time of excision. Consequently, assessment of the state of these kinase networks requires stabilization, or preservation, of the phosphoproteins immediately post tissue procurement. We have employed reverse phase protein microarray analysis of phosphoproteins to study the factors influencing stability of phosphoproteins in tissue following procurement. Based on this analysis we have established tissue procurement guidelines for clinical research with an emphasis on quantifying phosphoproteins by RPMA. PMID:21901591

  13. 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.

  14. Screening of differentially expressed genes in pathological scar tissues using expression microarray.

    PubMed

    Huang, L P; Mao, Z; Zhang, L; Liu, X X; Huang, C; Jia, Z S

    2015-01-01

    Pathological scar tissues and normal skin tissues were differentiated by screening for differentially expressed genes in pathologic scar tissues via gene expression microarray. The differentially expressed gene data was analyzed by gene ontology and pathway analyses. There were 5001 up- or down-regulated genes in 2-fold differentially expressed genes, 956 up- or down-regulated genes in 5-fold differentially expressed genes, and 114 up- or down-regulated genes in 20-fold differentially expressed genes. Therefore, significant differences were observed in the gene expression in pathological scar tissues and normal foreskin tissues. The development of pathological scar tissues has been correlated to changes in multiple genes and pathways, which are believed to form a dynamic network connection.

  15. Web-based tissue microarray image data analysis: initial validation testing through prostate cancer Gleason grading.

    PubMed

    Bova, G S; Parmigiani, G; Epstein, J I; Wheeler, T; Mucci, N R; Rubin, M A

    2001-04-01

    Tissue microarray technology promises to enhance tissue-based molecular research by allowing improved conservation of tissue resources and experimental reagents, improved internal experimental control, and increased sample numbers per experiment. Organized, well-validated collection and analysis of the voluminous image data produced by tissue microarray technology is critical to maximize its value. Web-based technology for visual analysis and searchable storage of microarray image data could provide optimal flexibility for research groups in meeting this goal, but this approach has not been examined scientifically. Toward this goal, a prostate tissue microarray block containing 432 tissue cores (0.6 mm diameter) was constructed. Moderately compressed (200 kb).jpg images of each tissue spot were acquired and were saved using a naming convention developed by the SPORE Prostate Tissue Microarray Collaborative Group. Four hundred three tissue array spot images were uploaded into a database developed for this study and were converted to.fpx format to decrease Internet transmission times for high-resolution image data. In phase I of the image analysis portion of the study, testing and preliminary analysis of the Web technology was performed by 2 pathologists (M.A.R. and G.S.B.). In phase II, 2 pathologists (J.I.E. and T.M.W.) with no previous exposure to this technology and no knowledge of the structure of the study were presented a set of 130 sequential tissue spot images via the Web on their office computers. In phase III, the same pathologists were presented a set of 193 images, including all 130 from phase II and 63 others, with image presentation order randomized. With each zoomable tissue spot image, each pathologist was presented with a nested set of questions regarding overall interpretability of the image, presence or absence of cancer, and predominant and second most frequent Gleason grade. In phases II and III of the study, 319 of 323 (99%) image presentations

  16. Progress of science from microscopy to microarrays (part 1): diagnosis of parasitic diseases.

    PubMed

    Dey, Ayan; Singh, Sarman

    2009-01-01

    Even though description of the magnifying glass goes back to 1021 by an Arabic physicist in his book, Antony van Leeuwenhoek was the first man to improve the then simple microscope for viewing biological specimens in 1674. This suggests that every discovery has scope for improvement, be it physics or be it biology. In the field of biology, scientists have long studied gene expression as a hallmark of gene activities reflecting the current cell conditions and response to host immune defense systems. These studies have been cumbersome, technically demanding and time-consuming. Application of microarrays has revolutionized this field and help understand the simultaneous expression of thousands of genes in a single sample put onto a single solid support. It is also now possible to compare gene expression in two different cell types, different stages of life cycle or two tissue samples, such as in healthy and diseased ones. Thus microarrays are beginning to dominate other conventional and molecular diagnostic technologies. The microarrays consist of solid supports onto which the nucleic acid sequences from thousands of different genes are immobilized, or attached at fixed locations. These solid supports themselves are usually glass slides, silicon chips or nylon membranes. The nucleic acids are spotted or synthesized directly onto the support. Application of microarrays is new for parasites. Most of these applications are done for monitoring parasite gene expression, to predict the functions of uncharacterized genes, probe the physiologic adaptations made under various environmental conditions, identify virulence-associated genes and test the effects of drug targets. The best examples are vector-borne parasites, such as Plasmodium, Trypanosoma and Leishmania, in which genes expressed, during mammalian and insect host stages, have been elucidated. Microarrays have also been successfully applied to understand the factors responsible to induce transformation from

  17. Expression of SATB1 protein in the ductal breast carcinoma tissue microarrays - preliminary study.

    PubMed

    Kobierzycki, Christopher; Wojnar, Andrzej; Dziegiel, Piotr

    2013-01-01

    Special AT-rich sequence-binding protein 1 (SATB1) is a nuclear matrix protein which interacts with specific regions of DNA, ensuring its proper organization and function in the cell. The expression of SATB1 was primarily found in thymocytes, but its increased levels were observed in various types of cancers. However, the knowledge of the function and application possibilities of this protein is still limited. The aim of this study was to investigate the expression of SATB1 protein using immunohistochemistry and tissue microarray (TMA) technique and determine its possible relationship with the proliferative marker Ki-67, estrogen a (ER) and progesterone (PR) receptors as well as grade of histological malignancy (G). The study was performed on material of 48 archival invasive ductal breast cancers (IDC). The TMAs were prepared with the use of 0.6 mm diameter punches. Immunohistochemical reactions were carried out using antibodies against Ki-67, ER, PR and SATB1 proteins. The intensity of the nuclear reaction was evaluated using a light microscope and computer-assisted image analysis. Expression of Ki-67 and SATB1 protein was observed in 89.58% and 31.25% of cancer cases, respectively. 62.5% of tumors were classified as ER-positive, and 47.92% as PR-positive. Statistical analysis showed a moderate positive correlation between Ki-67 and SATB1 expression (r = 0.291, p = 0.045 independently on the receptor status, and r = 0.392, p = 0.032 in ER-negative tumors). The expression of the Ki-67 antigen increased with higher grade of histological malignancy (G). The results suggest that SATB1 protein may play an indirect role in the cell proliferation and should be evaluated in relation to the other markers. Further studies concerning determination of its role in cancer progression and metastasis, in terms of application as therapeutic target and prognostic marker, are recommended.

  18. Identification of novel pancreatic adenocarcinoma cell-surface targets by gene expression profiling and tissue microarray.

    PubMed

    Morse, David L; Balagurunathan, Yoga; Hostetter, Galen; Trissal, Maria; Tafreshi, Narges K; Burke, Nancy; Lloyd, Mark; Enkemann, Steven; Coppola, Domenico; Hruby, Victor J; Gillies, Robert J; Han, Haiyong

    2010-09-01

    Pancreatic cancer has a high mortality rate, which is generally related to the initial diagnosis coming at late stage disease combined with a lack of effective treatment options. Novel agents that selectively detect pancreatic cancer have potential for use in the molecular imaging of cancer, allowing for non-invasive determination of tumor therapeutic response and molecular characterization of the disease. Such agents may also be used for the targeted delivery of therapy to tumor cells while decreasing systemic effects. Using complementary assays of mRNA expression profiling to determine elevated expression in pancreatic cancer tissues relative to normal pancreas tissues, and validation of protein expression by immunohistochemistry on tissue microarray, we have identified cell-surface targets with potential for imaging and therapeutic agent development. Expression profiles of 2177 cell-surface genes for 28 pancreatic tumor specimens and 4 normal pancreas tissue samples were evaluated. Expression in normal tissues was evaluated using array data from 103 samples representing 28 organ sites as well as mining published data. One-hundred seventy unique targets were highly expressed in 2 or more of the pancreatic tumor specimens and were not expressed in the normal pancreas samples. Two targets (TLR2 and ABCC3) were further validated for protein expression by tissue microarray (TMA) based immunohistochemistry. These validated targets have potential for the development of diagnostic imaging and therapeutic agents for pancreatic cancer.

  19. 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

  20. Progress in free tissue transfer.

    PubMed

    O'Brien, B M; Kumar, P A

    1990-01-01

    The development of microsurgical techniques has supplied plastic surgery with a new chance to transfer tissue to nearly any recipient site of the body. Classical methods still have their value but also their limits in many circumstances. Free tissue transfer has proved its advantages, especially in covering defects in the lower extremity; but microsurgical flaps also gain ground in reconstructive surgery of the head and neck, the female breast, the abdominal wall, and the hands. This article will survey well-classified microsurgical flaps, their indications, and their limits. In addition to a description of skin, fasciocutaneous, septocutaneous, and musculocutaneous flaps, we also include the latest detailed knowledge of free transfer of muscles, vascularized tendons, nerves, bones, and even joints, digits, and jejunal transfer. An outlook toward new perspectives with so-called prefabricated flaps will also be discussed. PMID:2256350

  1. Decentralized Data Sharing of Tissue Microarrays for Investigative Research in Oncology

    PubMed Central

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

    2007-01-01

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

  2. Microarray analysis of the AHR system: Tissue-specific flexibility in signal and target genes

    SciTech Connect

    Frericks, Markus; Meissner, Marc; Esser, Charlotte . E-mail: chesser@uni-duesseldorf.de

    2007-05-01

    Data mining published microarray experiments require that expression profiles are directly comparable. We performed linear global normalization on the data of 1967 Affymetrix U74av2 microarrays, i.e. the transcriptomes of > 100 murine tissues or cell types. The mathematical transformation effectively nullifies inter-experimental or inter-laboratory differences between microarrays. The correctness of expression values was validated by quantitative RT-PCR. Using the database we analyze components of the aryl hydrocarbon receptor (AHR) signaling pathway in various tissues. We identified lineage and differentiation specific variant expression of AHR, ARNT, and HIF1{alpha} in the T-cell lineage and high expression of CYP1A1 in immature B cells and dendritic cells. Performing co-expression analysis we found unorthodox expression of the AHR in the absence of ARNT, particularly in stem cell populations, and can reject the hypothesis that ARNT2 takes over and is highly expressed when ARNT expression is low or absent. Furthermore the AHR shows no co-expression with any other transcript present on the chip. Analysis of differential gene expression under 308 conditions revealed 53 conditions under which the AHR is regulated, numerous conditions under which an intrinsic AHR action is modified as well as conditions activating the AHR even in the absence of known AHR ligands. Thus meta-analysis of published expression profiles is a powerful tool to gain novel insights into known and unknown systems.

  3. Rapid microarray-based genotyping of Chlamydia spp. strains from clinical tissue samples.

    PubMed

    Sachse, Konrad; Ruettger, Anke

    2015-01-01

    Pathogenic Chlamydia (C.) psittaci and C. trachomatis strains can be genotyped based on variations in the ompA genomic locus. In the present chapter, we describe rapid genotyping assays for both chlamydial agents using the ArrayStrip™ (AS) microarray platform. The test is targeting multiple discriminatory sites in the variable domains of the ompA gene by using 35 (C. psittaci) and 61 (C. trachomatis) oligonucleotide probes representing genotype-specific polymorphisms. In addition to discrimination among the established genotypes, this approach allows identification of atypical strains that were not accessible to typing using previously established techniques, such as PCR-RFLP or serotyping. The present DNA microarray assay can be conducted directly on clinical tissue samples and is suitable for tracing epidemiological chains and exploring the dissemination of particular genotypes. The procedure is easy to handle and economically affordable, and it allows genotyping of up to 32 clinical samples per day, thus lending itself for routine diagnosis as well.

  4. A next-generation tissue microarray (ngTMA) protocol for biomarker studies.

    PubMed

    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

  5. Cancer and Leukemia Group B Pathology Committee Guidelines for Tissue Microarray Construction Representing Multicenter Prospective Clinical Trial Tissues

    PubMed Central

    Rimm, David L.; Nielsen, Torsten O.; Jewell, Scott D.; Rohrer, Daniel C.; Broadwater, Gloria; Waldman, Frederic; Mitchell, Kisha A.; Singh, Baljit; Tsongalis, Gregory J.; Frankel, Wendy L.; Magliocco, Anthony M.; Lara, Jonathan F.; Hsi, Eric D.; Bleiweiss, Ira J.; Badve, Sunil S.; Chen, Beiyun; Ravdin, Peter M.; Schilsky, Richard L.; Thor, Ann; Berry, Donald A.

    2011-01-01

    Practice-changing evidence requires confirmation, preferably in multi-institutional clinical trials. The collection of tissue within such trials has enabled biomarker studies and evaluation of companion diagnostic tests. Tissue microarrays (TMAs) have become a standard approach in many cooperative oncology groups. A principal goal is to maximize the number of assays with this precious tissue. However, production strategies for these arrays have not been standardized, possibly decreasing the value of the study. In this article, members of the Cancer and Leukemia Group B Pathology Committee relay our experiences as array facility directors and propose guidelines regarding the production of high-quality TMAs for cooperative group studies. We also discuss statistical issues arising from having a proportion of patients available for TMAs and the possibility that patients with TMAs fail to represent the greater study population. PMID:21519016

  6. 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.

  7. Microarray analysis on archival multiple sclerosis tissue: pathogenic authenticity outweighs technical obstacles.

    PubMed

    Bradl, Monika; Lassmann, Hans

    2012-08-01

    Probably all neuropathologists know this dilemma: on the one hand, they have extremely precious archival material in their possession, which has been collected over many years from many different laboratories. Typically, this material is extremely well characterized, and often, it contains especially significant tissue specimens from unique cases. On the other hand, they face severe scepticism when they plan to use this archival material for large-scale gene expression studies by microarray analysis, since previous handling in the absence of RNA protection, prolonged storage at room temperature, and fixation with formaldehyde may dramatically reduce the amount of retrievable RNA. Fortunately, this dilemma can be solved. We give here examples from our own, multiple sclerosis-centered laboratory and explain why archival tissue might be more authentic for the disease process and might yield more information about the molecular and cellular substrates driving CNS inflammation in MS patients than more recently acquired tissues.

  8. Multi-tissue microarray analysis identifies a molecular signature of regeneration.

    PubMed

    Mercer, Sarah E; Cheng, Chia-Ho; Atkinson, Donald L; Krcmery, Jennifer; Guzman, Claudia E; Kent, David T; Zukor, Katherine; Marx, Kenneth A; Odelberg, Shannon J; Simon, Hans-Georg

    2012-01-01

    The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

  9. Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

    PubMed Central

    Mercer, Sarah E.; Cheng, Chia-Ho; Atkinson, Donald L.; Krcmery, Jennifer; Guzman, Claudia E.; Kent, David T.; Zukor, Katherine; Marx, Kenneth A.; Odelberg, Shannon J.; Simon, Hans-Georg

    2012-01-01

    The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine. PMID:23300656

  10. Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization

    PubMed Central

    Hanna, Jason A.; Wimberly, Hallie; Kumar, Salil; Slack, Frank; Agarwal, Seema; Rimm, David L.

    2013-01-01

    MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression. PMID:22482439

  11. Tissue Microarray Technology for Molecular Applications: Investigation of Cross-Contamination between Tissue Samples Obtained from the Same Punching Device

    PubMed Central

    Vassella, Erik; Galván, José A.; Zlobec, Inti

    2015-01-01

    Background: Tissue microarray (TMA) technology allows rapid visualization of molecular markers by immunohistochemistry and in situ hybridization. In addition, TMA instrumentation has the potential to assist in other applications: punches taken from donor blocks can be placed directly into tubes and used for nucleic acid analysis by PCR approaches. However, the question of possible cross-contamination between samples punched with the same device has frequently been raised but never addressed. Methods: Two experiments were performed. (1) A block from mycobacterium tuberculosis (TB) positivetissue and a second from an uninfected patient were aligned side-by-side in an automated tissue microarrayer. Four 0.6 mm punches were cored from each sample and placed inside their corresponding tube. Between coring of each donor block, a mechanical cleaning step was performed by insertion of the puncher into a paraffin block. This sequence of coring and cleaning was repeated three times, alternating between positive and negative blocks. A fragment from the 6110 insertion sequence specific for mycobacterium tuberculosis was analyzed; (2) Four 0.6 mm punches were cored from three KRAS mutated colorectal cancer blocks, alternating with three different wild-type tissues using the same TMA instrument (sequence of coring: G12D, WT, G12V, WT, G13D and WT). Mechanical cleaning of the device between each donor block was made. Mutation analysis by pyrosequencing was carried out. This sequence of coring was repeated manually without any cleaning step between blocks. Results/Discussion: In both analyses, all alternating samples showed the expected result (samples 1, 3 and 5: positive or mutated, samples 2, 4 and 6: negative or wild-type). Similar results were obtained without cleaning step. These findings suggest that no cross-contamination of tissue samples occurs when donor blocks are punched using the same device, however a cleaning step is nonetheless recommended. Our result supports

  12. Microarray Based Gene Expression Analysis of Murine Brown and Subcutaneous Adipose Tissue: Significance with Human

    PubMed Central

    Boparai, Ravneet K.; Kondepudi, Kanthi Kiran; Mantri, Shrikant; Bishnoi, Mahendra

    2015-01-01

    Background Two types of adipose tissues, white (WAT) and brown (BAT) are found in mammals. Increasingly novel strategies are being proposed for the treatment of obesity and its associated complications by altering amount and/or activity of BAT using mouse models. Methodology/Principle Findings The present study was designed to: (a) investigate the differential expression of genes in LACA mice subcutaneous WAT (sWAT) and BAT using mouse DNA microarray, (b) to compare mouse differential gene expression with previously published human data; to understand any inter- species differences between the two and (c) to make a comparative assessment with C57BL/6 mouse strain. In mouse microarray studies, over 7003, 1176 and 401 probe sets showed more than two-fold, five-fold and ten-fold change respectively in differential expression between murine BAT and WAT. Microarray data was validated using quantitative RT-PCR of key genes showing high expression in BAT (Fabp3, Ucp1, Slc27a1) and sWAT (Ms4a1, H2-Ob, Bank1) or showing relatively low expression in BAT (Pgk1, Cox6b1) and sWAT (Slc20a1, Cd74). Multi-omic pathway analysis was employed to understand possible links between the organisms. When murine two fold data was compared with published human BAT and sWAT data, 90 genes showed parallel differential expression in both mouse and human. Out of these 90 genes, 46 showed same pattern of differential expression whereas the pattern was opposite for the remaining 44 genes. Based on our microarray results and its comparison with human data, we were able to identify genes (targets) (a) which can be studied in mouse model systems to extrapolate results to human (b) where caution should be exercised before extrapolation of murine data to human. Conclusion Our study provides evidence for inter species (mouse vs human) differences in differential gene expression between sWAT and BAT. Critical understanding of this data may help in development of novel ways to engineer one form of adipose

  13. MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

    PubMed

    Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

    2014-01-01

    A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

  14. Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays

    PubMed Central

    Sill, Martin; Schröder, Christoph; Shen, Ying; Marzoq, Aseel; Komel, Radovan; Hoheisel, Jörg D.; Nienhüser, Henrik; Schmidt, Thomas; Kastelic, Damjana

    2016-01-01

    In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL‐10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins’ capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches. PMID:27600085

  15. Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays.

    PubMed

    Sill, Martin; Schröder, Christoph; Shen, Ying; Marzoq, Aseel; Komel, Radovan; Hoheisel, Jörg D; Nienhüser, Henrik; Schmidt, Thomas; Kastelic, Damjana

    2016-01-01

    In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL-10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins' capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches. PMID:27600085

  16. Association between FOXM1 and hedgehog signaling pathway in human cervical carcinoma by tissue microarray analysis

    PubMed Central

    Chen, Hong; Wang, Jingjing; Yang, Hong; Chen, Dan; Li, Panpan

    2016-01-01

    Forkhead box M1 (FOXM1) and hedgehog (Hh) signaling pathway are implicated in the formation and development of human tumors, including cervical cancer. Previous studies have indicated that FOXM1 may be a downstream target gene of the Hh signaling pathway, but their association in cervical cancer is largely unknown. In the present study, the expression of FOXM1 and Hh signaling molecules was evaluated by immunohistochemical analysis in a tissue microarray that contained 70 cervical cancer tissues and 10 normal cervical tissues. In addition, the association of these molecules with clinicopathological parameters, and the association between FOXM1 and various molecules involved in the Hh signaling pathway was investigated. The results indicated that FOXM1 and Hh signaling molecules were overexpressed in cervical cancer tissues. The protein expression levels of FOXM1, glioma-associated oncogene 1 (GLI1) and smoothened (SMO) correlated with the clinical stage of the tumors, while the protein expression levels of Sonic Hh (SHh), patched 1 (PTCH1) and GLI1 correlated with the pathological grade of the tumors. The expression levels of GLI1 were lower in tissues without lymph node metastasis than in tissues with lymph node metastasis. In addition, FOXM1 expression correlated with GLI1, SHh and PTCH1 expression in cancer tissues. These findings confirmed the participation of FOXM1 and the Hh signaling pathway in cervical cancer. Furthermore, the finding that FOXM1 may be a downstream target gene of the Hh signaling pathway in cervical cancer provides a potential novel diagnostic and therapeutic target for cervical cancer.

  17. Association between FOXM1 and hedgehog signaling pathway in human cervical carcinoma by tissue microarray analysis

    PubMed Central

    Chen, Hong; Wang, Jingjing; Yang, Hong; Chen, Dan; Li, Panpan

    2016-01-01

    Forkhead box M1 (FOXM1) and hedgehog (Hh) signaling pathway are implicated in the formation and development of human tumors, including cervical cancer. Previous studies have indicated that FOXM1 may be a downstream target gene of the Hh signaling pathway, but their association in cervical cancer is largely unknown. In the present study, the expression of FOXM1 and Hh signaling molecules was evaluated by immunohistochemical analysis in a tissue microarray that contained 70 cervical cancer tissues and 10 normal cervical tissues. In addition, the association of these molecules with clinicopathological parameters, and the association between FOXM1 and various molecules involved in the Hh signaling pathway was investigated. The results indicated that FOXM1 and Hh signaling molecules were overexpressed in cervical cancer tissues. The protein expression levels of FOXM1, glioma-associated oncogene 1 (GLI1) and smoothened (SMO) correlated with the clinical stage of the tumors, while the protein expression levels of Sonic Hh (SHh), patched 1 (PTCH1) and GLI1 correlated with the pathological grade of the tumors. The expression levels of GLI1 were lower in tissues without lymph node metastasis than in tissues with lymph node metastasis. In addition, FOXM1 expression correlated with GLI1, SHh and PTCH1 expression in cancer tissues. These findings confirmed the participation of FOXM1 and the Hh signaling pathway in cervical cancer. Furthermore, the finding that FOXM1 may be a downstream target gene of the Hh signaling pathway in cervical cancer provides a potential novel diagnostic and therapeutic target for cervical cancer. PMID:27698840

  18. Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

    PubMed Central

    Kampf, Caroline; Olsson, IngMarie; Ryberg, Urban; Sjöstedt, Evelina; Pontén, Fredrik

    2012-01-01

    The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins

  19. Construction of tissue microarrays from core needle biopsies - a systematic literature review.

    PubMed

    Albanghali, Mohammad; Green, Andrew; Rakha, Emad; Aleskandarany, Mohamed; Nolan, Chris; Ellis, Ian; Cheung, Kwok-Leung

    2016-02-01

    In some clinical circumstances, core needle biopsy (CNB) may be the only source of material from cancer tissue for diagnostic use. The volume of tissue available in a CNB is low, and opportunities for research use can therefore be limited. The tissue microarray (TMA) principle, if applied to the use of CNBs, could facilitate research studies in circumstances where CNB specimens are available. However, various challenges are expected in applying such a technique in CNBs, which has limited their use in research. We therefore conducted a systematic review of the literature on this subject. A systematic search was carried out with CINAHL, EMBASE, the Cochrane library, and MEDLINE, to identify studies that have primarily developed methods for constructing TMAs from CNBs. Eight studies were found to meet the inclusion criteria; six of these employed the vertical rearrangement technique, and two used multiple layers of biopsy tissue. Representation of the CNB was significantly influenced by the quantity of tumour cells present in the original biopsy and the degree of heterogeneity of biomarker expression. This review shows that technologies have been developed to enable construction of TMAs from CNBs. However, challenges remain to improve amplification and representation.

  20. Overview on Techniques to Construct Tissue Arrays with Special Emphasis on Tissue Microarrays

    PubMed Central

    Vogel, Ulrich

    2014-01-01

    With the advent of new histopathological staining techniques (histochemistry, immunohistochemistry, in situ hybridization) and the discovery of thousands of new genes, mRNA, and proteins by molecular biology, the need grew for a technique to compare many different cells or tissues on one slide in a cost effective manner and with the possibility to easily track the identity of each specimen: the tissue array (TA). Basically, a TA consists of at least two different specimens per slide. TAs differ in the kind of specimens, the number of specimens installed, the dimension of the specimens, the arrangement of the specimens, the embedding medium, the technique to prepare the specimens to be installed, and the technique to construct the TA itself. A TA can be constructed by arranging the tissue specimens in a mold and subsequently pouring the mold with the embedding medium of choice. In contrast, preformed so-called recipient blocks consisting of the embedding medium of choice have punched, drilled, or poured holes of different diameters and distances in which the cells or tissue biopsies will be deployed manually, semi-automatically, or automatically. The costs of constructing a TA differ from a few to thousands of Euros depending on the technique/equipment used. Remarkably high quality TAs can be also achieved by low cost techniques. PMID:27600339

  1. 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

  2. Distribution of AQP2 and AQP3 water channels in human tissue microarrays.

    PubMed

    Mobasheri, A; Wray, S; Marples, D

    2005-02-01

    The objective of this investigation was to use semi-quantitative immunohistochemistry to determine the distribution and expression levels of AQP2 and AQP3 proteins in normal human Tissue MicroArrays. Expression of the vasopressin regulated AQP2 was observed in a limited number of tissues. AQP2 was prominent in the apical and subapical plasma membranes of cortical and medullary renal collecting ducts. Surprisingly, weak AQP2 immunoreactivity was also noted in pancreatic islets, fallopian tubes and peripheral nerves. AQP2 was also localized to selected parts of the central nervous system (ependymal cell layer, subcortical white matter, hippocampus, spinal cord) and selected cells in the gastrointestinal system (antral and oxyntic gastric mucosa, small intestine and colon). These findings corroborate the restricted tissue distribution of AQP2. AQP3 was strongly expressed in many of the human tissues examined particularly in basolateral membranes of the distal nephron (medullary collecting ducts), distal colon, upper airway epithelia, transitional epithelium of the urinary bladder, tracheal, bronchial and nasopharyngeal epithelium, stratified squamous epithelial cells of the esophagus, and anus. AQP3 was moderately expressed in basolateral membranes of prostatic tubuloalveolar epithelium, pancreatic ducts, uterine endometrium, choroid plexus, articular chondrocytes, subchondral osteoblasts and synovium. Low AQP3 levels were also detected in skeletal muscle, cardiac muscle, gastric pits, seminiferous tubules, lymphoid vessels, salivary and endocrine glands, amniotic membranes, placenta and ovary. The abundance of basolateral AQP3 in epithelial tissues and its expression in many non-epithelial cells suggests that this aquaglyceroporin is a major participant in barrier hydration and water and osmolyte homeostasis in the human body. PMID:15703994

  3. Digital pathology for the validation of tissue microarrays in peripheral T-cell lymphomas.

    PubMed

    Pedersen, Martin B; Riber-Hansen, Rikke; Nielsen, Patricia S; Bendix, Knud; Hamilton-Dutoit, Stephen J; D'Amore, Francesco; Steiniche, Torben

    2014-09-01

    Systematic validation of construction and analysis parameters when using tissue microarray (TMA) in rare, morphologically heterogenous entities such as peripheral T-cell lymphoma (PTCL) is not reported. We describe a tissue-saving virtual TMA to predetermine the number of cores needed to represent whole tissue sections (WTS) from the same biopsies, using automated and traditional manual methods for the quantification of immunohistochemical stains. Whole paraffin hematoxylin and eosin- and immunohistochemical (CD2, CD30, and Ki-67)-stained sections from 30 PTCLs were digitalized. A virtual TMA with six 1-mm cores per slide was designed to compare agreements in the immunohistochemical scoring. Using digital image analysis and manual stereological counting, immunohistochemical positivity was quantified. Associations were analyzed using the Bland-Altman and correlation plots. In PTCL, we report that 4 cores are required to represent WTS results (ie, agreement within ±10%). High concordance was demonstrated between digital results obtained with WTS compared with 4-core virtual TMA (correlation coefficients: 0.89-0.98), and in the comparative evaluation of 4-core virtual TMA by digital image analysis versus manual stereology (correlation coefficients: 0.91 to 0.99). Virtual TMAs provide an efficient tool for optimizing and validating TMA construction parameters when planning a study. The method can be applied to the same tissues used in a subsequent formal study, without wasting scarce tissue resources. In PTCL, TMAs constructed with four 1-mm cores are representative of WTS. In parallel tests using TMAs and WTS from PTCLs, there is a high level of agreement comparing automated digital with manual stereological methods for the quantification of immunohistochemical biomarker staining.

  4. Validation of tissue microarray for molecular profiling of canine and feline mammary tumours.

    PubMed

    Muscatello, L V; Sarli, G; Beha, G; Asproni, P; Millanta, F; Poli, A; De Tolla, L J; Benazzi, C; Brunetti, B

    2015-01-01

    Tissue microarray (TMA) is a high-throughput method adopted for simultaneous molecular profiling of tissue samples from large patient cohorts. The aim of this study was to validate the TMA method for the molecular classification of canine and feline mammary tumours. Twelve samples, five feline and five canine mammary tumours and two canine haemangiosarcomas, were collected. TMA construction was based on Kononen's method of extracting a cylindrical core of paraffin wax-embedded 'donor' tissue and inserting it into a 'recipient' wax block. Seven consecutive sections from each tissue array block were subjected to immunohistochemistry (IHC) using primary antibodies specific for oestrogen receptor (OR), progesterone receptor (PR), c-erbB-2, cytokeratin (CK) 5/6, CK14, CK19 and p63. The same panel of antibodies was applied to the full sections from all cases. Comparison between full sections and TMA scores revealed different results depending on the antibodies. Labelling for OR, PR, CK19 and p63 showed total concordance, c-erbB2 (score +2, +3) was concordant in nine out of ten cases, CK5/6 and CK14 in eight out of ten cases. The TMA platform preserves the molecular profile of canine and feline mammary tumour markers, representing a useful tool for rapid and cost-effective analysis for the first phenotypic screening using OR, PR and c-erbB2 antibodies. Basal cytokeratin, used for triple negative identification, shows a multifocal 'niche' expression pattern, for which IHC of the full section or multiple core array is recommended. PMID:25670670

  5. Identification of New Players in Hepatocarcinogenesis: Limits and Opportunities of Using Tissue Microarray (TMA)

    PubMed Central

    Quagliata, Luca; Schlageter, Manuel; Quintavalle, Cristina; Tornillo, Luigi; Terracciano, Luigi M.

    2014-01-01

    Liver tumours are among the leading causes of cancer-related death worldwide and hepatocellular carcinoma (HCC) accounts for the vast majority of liver tumours. When detected at an early stage of disease, patients might still be eligible for surgical-based curative treatments. However, currently only small portion of HCC affected patients are diagnosed at an early stage. For late stage HCC no treatment option exists beside the multi-tyrosine kinase inhibitor Sorafenib. Thus new molecular targets and treatment options for HCC are urgently needed. Nevertheless, despite some improvements in diagnosis and patient management, the biology of liver tumour remains inadequately understood, mainly because these tumours have shown to harbour a highly complex genomic landscape. In addition, one major obstacle delaying the identification of new molecular targets in biomedical research is the necessity to validate them using a large collection of tissue specimens. Tissue microarray (TMA) technology allows the prompt molecular profiling of multiple tissue specimens and is therefore ideal to analyze presumptive candidate biomarkers in a fast an effective manner. The use of TMA has substantial benefits over standard techniques and represents a significant advancement in molecular pathology. For example, TMA technology reduces laboratory work, offers a high level of experimental uniformity and provides a judicious use of precious tissue. On the other hand, one potential limitation of using TMA is that the small cores sampled may not be representative of whole tumors. This issue is very critical in particularly heterogeneous cancers such as HCC. For liver focused studies, it is ideal to evaluate the staining patters of a determined marker over the structure of an entire acinus and to define staining in as many as possible anatomical regions. In this review we analyze the limits and opportunities offered by the usage of TMA technology in HCC research. In summary, TMA has

  6. Deciphering signaling pathways in clinical tissues for personalized medicine using protein microarrays.

    PubMed

    Malinowsky, K; Wolff, C; Ergin, B; Berg, D; Becker, K F

    2010-11-01

    The current transition in cancer therapy from general treatment approaches, based mainly on chemotherapy and radiotherapy, to more directed approaches that aim to inhibit specific molecular targets has brought about new challenges for pathology. In the past, classical assignment of pathology consisted of tumor diagnosis and staging for further therapy decisions; nowadays, pathologists are asked to predict possible therapeutic results by detecting and quantifying therapeutic targets in tumors such as the human epidermal growth factor receptor 2 (HER2). The best approach to analyze such molecular targets is to provide a tumor-specific protein expression profile prior to therapy. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow fast, precise, cheap, and simultaneous analysis of many network components while requiring only a small amount of clinical material. Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling quantitative measurement of proteins. Recently, methods for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues have become available. In this article, we demonstrate how the use of RPPA to analyze signaling pathways from FFPE tissues may improve quantification of therapeutic targets and diagnostic markers in the near future.

  7. Biomarkers for Refractory Lupus Nephritis: A Microarray Study of Kidney Tissue.

    PubMed

    Benjachat, Thitima; Tongyoo, Pumipat; Tantivitayakul, Pornpen; Somparn, Poorichaya; Hirankarn, Nattiya; Prom-On, Santitham; Pisitkun, Prapaporn; Leelahavanichkul, Asada; Avihingsanon, Yingyos; Townamchai, Natavudh

    2015-06-23

    The prognosis of severe lupus nephritis (LN) is very different among individual patients. None of the current biomarkers can be used to predict the development of refractory LN. Because kidney histology is the gold standard for diagnosing LN, the authors hypothesize that molecular signatures detected in kidney biopsy tissue may have predictive value in determining the therapeutic response. Sixty-seven patients with biopsy-proven severely active LN by International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification III/IV were recruited. Twenty-three kidney tissue samples were used for RNA microarray analysis, while the remaining 44 samples were used for validation by real-time polymerase chain reaction (PCR) gene expression analysis. From hundreds of differential gene expressions in refractory LN, 12 candidates were selected for validation based on gene expression levels as well as relevant functions. The candidate biomarkers were members of the innate immune response molecules, adhesion molecules, calcium-binding receptors, and paracellular tight junction proteins. S100A8, ANXA13, CLDN19 and FAM46B were identified as the best kidney biomarkers for refractory LN, and COL8A1 was identified as the best marker for early loss of kidney function. These new molecular markers can be used to predict refractory LN and may eventually lead to novel molecular targets for therapy.

  8. 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.

  9. Biomarkers for Refractory Lupus Nephritis: A Microarray Study of Kidney Tissue

    PubMed Central

    Benjachat, Thitima; Tongyoo, Pumipat; Tantivitayakul, Pornpen; Somparn, Poorichaya; Hirankarn, Nattiya; Prom-On, Santitham; Pisitkun, Prapaporn; Leelahavanichkul, Asada; Avihingsanon, Yingyos; Townamchai, Natavudh

    2015-01-01

    The prognosis of severe lupus nephritis (LN) is very different among individual patients. None of the current biomarkers can be used to predict the development of refractory LN. Because kidney histology is the gold standard for diagnosing LN, the authors hypothesize that molecular signatures detected in kidney biopsy tissue may have predictive value in determining the therapeutic response. Sixty-seven patients with biopsy-proven severely active LN by International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification III/IV were recruited. Twenty-three kidney tissue samples were used for RNA microarray analysis, while the remaining 44 samples were used for validation by real-time polymerase chain reaction (PCR) gene expression analysis. From hundreds of differential gene expressions in refractory LN, 12 candidates were selected for validation based on gene expression levels as well as relevant functions. The candidate biomarkers were members of the innate immune response molecules, adhesion molecules, calcium-binding receptors, and paracellular tight junction proteins. S100A8, ANXA13, CLDN19 and FAM46B were identified as the best kidney biomarkers for refractory LN, and COL8A1 was identified as the best marker for early loss of kidney function. These new molecular markers can be used to predict refractory LN and may eventually lead to novel molecular targets for therapy. PMID:26110394

  10. Microarray Expression Data Identify DCC as a Candidate Gene for Early Meningioma Progression

    PubMed Central

    Schulten, Hans-Juergen; Hussein, Deema; Al-Adwani, Fatima; Karim, Sajjad; Al-Maghrabi, Jaudah; Al-Sharif, Mona; Jamal, Awatif; Al-Ghamdi, Fahad; Baeesa, Saleh S.; Bangash, Mohammed; Chaudhary, Adeel; Al-Qahtani, Mohammed

    2016-01-01

    Meningiomas are the most common primary brain tumors bearing in a minority of cases an aggressive phenotype. Although meningiomas are stratified according to their histology and clinical behavior, the underlying molecular genetics predicting aggressiveness are not thoroughly understood. We performed whole transcript expression profiling in 10 grade I and four grade II meningiomas, three of which invaded the brain. Microarray expression analysis identified deleted in colorectal cancer (DCC) as a differentially expressed gene (DEG) enabling us to cluster meningiomas into DCC low expression (3 grade I and 3 grade II tumors), DCC medium expression (2 grade I and 1 grade II tumors), and DCC high expression (5 grade I tumors) groups. Comparison between the DCC low expression and DCC high expression groups resulted in 416 DEGs (p-value < 0.05; fold change > 2). The most significantly downregulated genes in the DCC low expression group comprised DCC, phosphodiesterase 1C (PDE1C), calmodulin-dependent 70kDa olfactomedin 2 (OLFM2), glutathione S-transferase mu 5 (GSTM5), phosphotyrosine interaction domain containing 1 (PID1), sema domain, transmembrane domain (TM) and cytoplasmic domain, (semaphorin) 6D (SEMA6D), and indolethylamine N-methyltransferase (INMT). The most significantly upregulated genes comprised chromosome 5 open reading frame 63 (C5orf63), homeodomain interacting protein kinase 2 (HIPK2), and basic helix-loop-helix family, member e40 (BHLHE40). Biofunctional analysis identified as predicted top upstream regulators beta-estradiol, TGFB1, Tgf beta complex, LY294002, and dexamethasone and as predicted top regulator effectors NFkB, PIK3R1, and CREBBP. The microarray expression data served also for a comparison between meningiomas from female and male patients and for a comparison between brain invasive and non-invasive meningiomas resulting in a number of significant DEGs and related biofunctions. In conclusion, based on its expression levels, DCC may constitute

  11. Quantitative analysis of p53 expression in human normal and cancer tissue microarray with global normalization method

    PubMed Central

    Idikio, Halliday A

    2011-01-01

    Tissue microarray based immunohistochemical staining and proteomics are important tools to create and validate clinically relevant cancer biomarkers. Immunohistochemical stains using formalin-fixed tissue microarray sections for protein expression are scored manually and semi-quantitatively. Digital image analysis methods remove some of the drawbacks of manual scoring but may need other methods such as normalization to provide across the board utility. In the present study, quantitative proteomics-based global normalization method was used to evaluate its utility in the analysis of p53 protein expression in mixed human normal and cancer tissue microarray. Global normalization used the mean or median of β-actin to calculate ratios of individual core stain intensities, then log transformed the ratios, calculate a mean or median and subtracted the value from the log of ratios. In the absence of global normalization of p53 protein expression, 44% (42 of 95) of tissue cores were positive using the median of intensity values and 40% (38 of 95) using the mean of intensities as cut-off points. With global normalization, p53 positive cores changed to 20% (19 of 95) when using median of intensities and 15.8%(15 of 95) when the mean of intensities were used. In conclusion, the global normalization method helped to define positive p53 staining in the tissue microarray set used. The method used helped to define clear cut-off points and confirmed all negatively stained tissue cores. Such normalization methods should help to better define clinically useful biomarkers. PMID:21738821

  12. Selective Invocation of Shape Priors for Deformable Segmentation and Morphologic Classification of Prostate Cancer Tissue Microarrays

    PubMed Central

    Ali, Sahirzeeshan; Veltri, Robert; Epstein, Jonathan A.; Christudass, Christhunesa; Madabhushi, Anant

    2015-01-01

    Shape based active contours have emerged as a natural solution to overlap resolution. However, most of these shape-based methods are computationally expensive. There are instances in an image where no overlapping objects are present and applying these schemes results in significant computational overhead without any accompanying, additional benefit. In this paper we present a novel adaptive active contour scheme (AdACM) that combines boundary and region based energy terms with a shape prior in a multi level set formulation. To reduce the computational overhead, the shape prior term in the variational formulation is only invoked for those instances in the image where overlaps between objects are identified; these overlaps being identified via a contour concavity detection scheme. By not having to invoke all 3 terms (shape, boundary, region) for segmenting every object in the scene, the computational expense of the integrated active contour model is dramatically reduced, a particularly relevant consideration when multiple objects have to be segmented on very large histopathological images. The AdACM was employed for the task of segmenting nuclei on 80 prostate cancer tissue microarray images from 40 patient studies. Nuclear shape based, architectural and textural features extracted from these segmentations were extracted and found to able to discriminate different Gleason grade patterns with a classification accuracy of 86% via a quadratic discriminant analysis (QDA) classifier. On average the AdACM model provided 60% savings in computational times compared to a non-optimized hybrid active contour model involving a shape prior. PMID:25466771

  13. Tissue microarray-based study of hepatocellular carcinoma validating SPIB as potential clinical prognostic marker.

    PubMed

    Ho, Yi-Jung; Lin, Yueh-Min; Huang, Yen-Chi; Yeh, Kun-Tu; Lin, Liang-In; Lu, Jeng-Wei

    2016-01-01

    Currently, the prognostic significance of SPIB protein overexpression in human hepatocellular carcinoma (HCC) is unclear. The aim of the present study was to investigate the level of SPIB expression in human HCC in order to determine possible correlations between SPIB expression and clinicopathological findings. The expression of SPIB proteins was detected using immunohistochemical staining in commercial multiple-tissue microarrays as a means of examining expression profiles in patients. Using online biomarker validation tool SurvExpress, we focused on the correlation between SPIB overexpression and survival as well as relapse-free survival (RFS). Results show that SPIB protein expression levels were significantly higher in colon, liver, and stomach tumors than in non-tumor tissues (p<0.05). SPIB overexpression in patients with HCC was also significantly higher than that of the normal samples (p<0.001). Among patients with liver disease, SPIB protein expression levels differ significantly according to the stage of liver disease, specifically between stages I, II, and III of HCC (p<0.05). SPIB expression was also shown to be significantly correlated with age (p=0.046) and histological grade (p=0.027). Furthermore, the SurvExpress analysis suggested that high SPIB and KI-67 mRNA expression were significantly associated with the poor survival of patients with HCC (p<0.05). Our results indicate that cross-talk in the expression of SPIB and KI-67 may be associated with poor prognosis and may potentially serve as a clinical prognostic indicator of HCC. This is the first time that such an association has been reported. PMID:26610895

  14. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues.

    PubMed

    García-Chequer, A J; Méndez-Tenorio, A; Olguín-López, G; Sánchez-Vallejo, C; Isa, P; Arias, C F; Torres, J; Hernández-Angeles, A; Ramírez-Ortiz, M A; Lara, C; Cabrera-Muñoz, Ma de L; Sadowinski-Pine, S; Bravo-Ortiz, J C; Ramón-García, G; Diegopérez-Ramírez, J; Ramírez-Reyes, G; Casarrubias-Islas, R; Ramírez, J; Orjuela, M; Ponce-Castañeda, M V

    2016-03-01

    Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma.

  15. Tissue microarray immunohistochemical profiles of p53 and pRB in hepatocellular carcinoma and hepatoblastoma.

    PubMed

    Azlin, Abdul Hadi; Looi, Lai Meng; Cheah, Phaik Leng

    2014-01-01

    The tumour suppressor genes, p53 and pRb, are known to play important roles in neoplastic transformation. While molecular routes to the uncontrolled growth of hepatocytes, leading to primary liver cancer have generated considerable interest, the roles of p53 and pRb mutations in hepatocellular carcinoma (HCC) and hepatoblastoma (HB) remain to be clarified. We examined the immunohistochemical expression of p53 and pRb gene products in 26 HCC and 9 HB, sampled into tissue microarray blocks. 10 (38%) of 26 HCC showed > 10% tumour nuclear staining for p53 protein, 3 of these also being HbsAg positive. Conversely, none of 9 HB expressed nuclear p53 immunopositivity. Some 24 (92%) HCC and 8 (89%) HB showed loss of pRb nuclear expression. Two of the 26 HCC and one of the 9 HB showed >10% tumour nuclear staining for pRb protein. Our results suggest that p53 does not have an important role in the development of HB but may contribute in HCC. There is also loss of pRb expression in the majority of HCC and HB, supporting loss of pRb gene function in the hepatocarcinogenesis pathway. However, a comparison of the staining profiles of p53 and pRb proteins in HCC and HB did not reveal a consistent pattern to differentiate between the two types of tumours immunohistochemically. Hence the use of p53 and pRB protein expression has no contribution in the situation where there is a diagnostic difficulty in deciding between HCC and HB.

  16. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues

    PubMed Central

    García-Chequer, A.J.; Méndez-Tenorio, A.; Olguín-López, G.; Sánchez-Vallejo, C.; Isa, P.; Arias, C.F.; Torres, J.; Hernández-Angeles, A.; Ramírez-Ortiz, M.A.; Lara, C.; Cabrera-Muñoz, Ma.de.L.; Sadowinski-Pine, S.; Bravo-Ortiz, J.C.; Ramón-García, G.; Diegopérez-Ramírez, J.; Ramírez-Reyes, G.; Casarrubias-Islas, R.; Ramírez, J.; Orjuela, M.; Ponce-Castañeda, M.V.

    2016-01-01

    Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma. PMID:26937470

  17. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues.

    PubMed

    García-Chequer, A J; Méndez-Tenorio, A; Olguín-López, G; Sánchez-Vallejo, C; Isa, P; Arias, C F; Torres, J; Hernández-Angeles, A; Ramírez-Ortiz, M A; Lara, C; Cabrera-Muñoz, Ma de L; Sadowinski-Pine, S; Bravo-Ortiz, J C; Ramón-García, G; Diegopérez-Ramírez, J; Ramírez-Reyes, G; Casarrubias-Islas, R; Ramírez, J; Orjuela, M; Ponce-Castañeda, M V

    2016-03-01

    Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at http://www.ebi.ac.uk/ena/data/view/PRJEB6630). We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at http://www.ncbi.nlm.nih.gov/geo/accession number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma. PMID:26937470

  18. Recent progress in tissue optical clearing

    PubMed Central

    Zhu, Dan; Larin, Kirill V; Luo, Qingming; Tuchin, Valery V

    2013-01-01

    Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This paper gives a review of recent developments in tissue optical clearing techniques. The physical, molecular and physiological mechanisms of tissue optical clearing are overviewed and discussed. Various methods for enhancing penetration of optical-clearing agents into tissue, such as physical methods, chemical-penetration enhancers and combination of physical and chemical methods are introduced. Combining the tissue optical clearing technique with advanced microscopy image or labeling technique, applications for 3D microstructure of whole tissues such as brain and central nervous system with unprecedented resolution are demonstrated. Moreover, the difference in diffusion and/or clearing ability of selected agents in healthy versus pathological tissues can provide a highly sensitive indicator of the tissue health/pathology condition. Finally, recent advances in optical clearing of soft or hard tissue for in vivo imaging and phototherapy are introduced. PMID:24348874

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

    PubMed

    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

  20. A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs

    PubMed Central

    Beuvink, Iwan; Kolb, Fabrice A.; Budach, Wolfgang; Garnier, Arlette; Lange, Joerg; Natt, Francois; Dengler, Uwe; Hall, Jonathan; Weiler, Jan

    2007-01-01

    Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2′-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity. PMID:17355992

  1. Expression and clinicopathological implication of DcR3 in lung cancer tissues: a tissue microarray study with 365 cases

    PubMed Central

    Zhang, Yu; Luo, Jie; He, Rongquan; Huang, Wenting; Li, Zuyun; Li, Ping; Dang, Yiwu; Chen, Gang; Li, Shikang

    2016-01-01

    Background Decoy receptor 3 (DcR3) has been reported to be involved in different cancers. However, few related researches have been accomplished on the role of DcR3 in lung cancer. Objective To explore the expression level and clinicopathological implication of DcR3 protein in lung cancer tissues. Materials and methods Immunohistochemistry was used to examine DcR3 protein expression in lung cancer (n=365) and normal lung tissues (n=26). The relationships between DcR3 expression and clinical parameters were further investigated. Furthermore, the diagnostic and clinicopathological value of DcR3 mRNA was analyzed based on The Cancer Genome Atlas database in lung cancer patients. Results Compared to normal lung tissues, DcR3 expression was significantly higher in lung cancer (P=0.007) tissues, including small-cell lung cancer (P=0.001) and non-small-cell lung cancer (P=0.008). In addition, DcR3 expression was related to tumor-node-metastasis (TNM) stage (P<0.001), tumor diameter (P=0.007), distant metastasis (P<0.001), and lymph node metastasis (P<0.001) in lung cancers. When concerning non-small-cell lung cancer, consistent correlations between DcR3 expression and TNM stage (P<0.001), tumor diameter (P=0.019), distant metastasis (P<0.001), and lymph node metastasis (P<0.001) were found. Simultaneously, in small-cell lung cancer, TNM stage (P=0.004) and lymph node metastasis (P=0.005) were also associated with DcR3 expression. Additionally, receiver operator characteristic curve revealed that the area under curve (AUC) of DcR3 was 0.637 (95% confidence interval [CI] 0.531–0.742) for lung cancer. Furthermore, DcR3 was overexpressed in both adenocarcinoma and squamous cell carcinoma tissues than in noncancerous lung tissues (all P<0.0001) based on the data from The Cancer Genome Atlas. AUC of DcR3 was 0.726 (95% CI 0.644–0.788) for lung adenocarcinoma patients and 0.647 (95% CI 0.566–0.728) for squamous cell carcinoma patients. DcR3 expression was also related to

  2. Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots.

    PubMed

    Au, Giang H T; Mejias, Linette; Swami, Vanlila K; Brooks, Ari D; Shih, Wan Y; Shih, Wei-Heng

    2014-03-01

    In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative. PMID:24411673

  3. [Protein microarrays and personalized medicine].

    PubMed

    Yu, Xiabo; Schneiderhan-Marra, Nicole; Joos, Thomas O

    2011-01-01

    Over the last 10 years, DNA microarrays have achieved a robust analytical performance, enabling their use for analyzing the whole transcriptome or for screening thousands of single-nucleotide polymorphisms in a single experiment. DNA microarrays allow scientists to correlate gene expression signatures with disease progression, to screen for disease-specific mutations, and to treat patients according to their individual genetic profiles; however, the real key is proteins and their manifold functions. It is necessary to achieve a greater understanding of not only protein function and abundance but also their role in the development of diseases. Protein concentrations have been shown to reflect the physiological and pathologic state of an organ, tissue, or cells far more directly than DNA, and proteins can be profiled effectively with protein microarrays, which require only a small amount of sample material. Protein microarrays have become wellestablished tools in basic and applied research, and the first products have already entered the in vitro diagnostics market. This review focuses on protein microarray applications for biomarker discovery and validation, disease diagnosis, and use within the area of personalized medicine. Protein microarrays have proved to be reliable research tools in screening for a multitude of parameters with only a minimal quantity of sample and have enormous potential in applications for diagnostic and personalized medicine.

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

    PubMed

    He, Fei; Yoo, Shinjae; Wang, Daifeng; Kumari, Sunita; Gerstein, Mark; Ware, Doreen; Maslov, Sergei

    2016-06-01

    Transcriptome data sets 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 a lack of metadata or differences in annotation styles of different labs. In this study, we carefully selected and integrated 6057 Arabidopsis microarray expression samples from 304 experiments deposited to the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI). Metadata such as tissue type, growth conditions and developmental stage were manually curated for each sample. We then studied the global expression landscape of the integrated data set and 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 with aerial tissues, but the transcriptome of cultured root is more similar to the transcriptome of aerial tissues, as the cultured root 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 the 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 the accuracy of our predictions with sample metadata provided by the authors. PMID:27015116

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

    DOE PAGES

    He, Fei; Maslov, Sergei; Yoo, Shinjae; Wang, Daifeng; Kumari, Sunita; Gerstein, Mark; Ware, Doreen

    2016-03-25

    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

  6. A 'waterfall' transfer-based workflow for improved quality of tissue microarray construction and processing in breast cancer research.

    PubMed

    Oberländer, M; Alkemade, H; Bünger, S; Ernst, F; Thorns, C; Braunschweig, T; Habermann, J K

    2014-07-01

    A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss. PMID:24619867

  7. A 'waterfall' transfer-based workflow for improved quality of tissue microarray construction and processing in breast cancer research.

    PubMed

    Oberländer, M; Alkemade, H; Bünger, S; Ernst, F; Thorns, C; Braunschweig, T; Habermann, J K

    2014-07-01

    A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss.

  8. [Study progress of dental pulp stem cells in tissue engineering].

    PubMed

    Shiyu, Shi; Jiamin, Xie

    2015-12-01

    In recent years, modern tissue engineering is becoming emerging and developing rapidly, and the acquisition, cultivation and differentiation of seed cells is the premise and foundation of the construction of tissue engineering, so more and more scholars pay attention to stem cells as seed cells for tissue engineering construction. Dental pulp stem cells (DPSCs) is a kind of adult stem cells derived from dental pulp, and as a new kind of seed cells of tissue engineering, the study of DPSCs presents important significance in tissue and organ regeneration. In this review, we introduced the progress of studies on dental pulp stem cells and discussed their clinical application prospects. PMID:27051964

  9. Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

    PubMed Central

    Callari, Maurizio; Dugo, Matteo; Musella, Valeria; Marchesi, Edoardo; Chiorino, Giovanna; Grand, Maurizia Mello; Pierotti, Marco Alessandro; Daidone, Maria Grazia; Canevari, Silvana; De Cecco, Loris

    2012-01-01

    Background Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples. Methodology/Principal Findings We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport. Conclusions Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach. PMID:23028787

  10. Study Progress on Tissue Culture of Maize Mature Embryo

    NASA Astrophysics Data System (ADS)

    Wang, Hongzhen; Cheng, Jun; Cheng, Yanping; Zhou, Xioafu

    It has been paid more and more attention on maize tissue culture as it is a basic work in maize genetic transformation, especially huge breakthrough has been made in maize tissue culture utilizing mature embryos as explants in the recent years. This paper reviewed the study progress on maize tissue culture and plant regeneration utilizing mature embryos as explants from callus induction, subculture, plant regeneration and browning reduction and so on.

  11. Heterogeneity of ERBB2 in gastric carcinomas: a study of tissue microarray and matched primary and metastatic carcinomas.

    PubMed

    Cho, Eun Yoon; Park, Kyeongmee; Do, Ingu; Cho, Junhun; Kim, Jiyun; Lee, Jeeyun; Kim, Seonwoo; Kim, Kyoung-Mee; Sohn, Tae Sung; Kang, Won Ki; Kim, Sung

    2013-05-01

    Trastuzumab in association with systemic cytotoxic chemotherapy is a therapeutic option for patients with advanced or metastatic ERBB2+ gastric carcinoma. The status of the ERBB2 overexpression or gene amplification is an important predictive marker in gastric cancer. However, it is controversial whether the primary tumor is representative of distant metastases in terms of ERBB2 status. Quadruplicated tissue microarrays from formalin-fixed paraffin-embedded tissues from 498 advanced primary gastric carcinomas and 97 matched metastatic lymph nodes were investigated by immunohistochemistry with HercepTest and silver in situ hybridization. For further comparison, another set of 41 paired primary and distant metastatic gastric carcinomas were also tested. Intratumoral heterogeneity was defined as different results between tissue microarray cores. ERBB2-positivity was observed in 52 gastric carcinomas (10%) and was not associated with recurrence of disease or survival of patients. In ERBB2-positive primary gastric carcinomas, heterogeneous ERBB2 overexpression was observed in 21/63 (33%) gastric carcinomas and heterogeneous ERBB2 gene amplification in 14/62 (23%) cases. Repeated immunohistochemistry and silver in situ hybridization in representative paraffin tumor blocks confirmed focal ERBB2 overexpression and ERBB2 gene amplification and did not change the final results. Discrepancies in ERBB2 results between primary and paired metastatic lymph nodes were observed in 11% of cases by immunohistochemistry and 7% by silver in situ hybridization. Out of the 41 paired primary and distant metastases, 5 (12%) cases were ERBB2-positive, and discrepancy was observed in one case. Intratumoral heterogeneity and discrepant ERBB2 results in primary and metastatic tumor are not uncommon in gastric carcinoma. Results of silver in situ hybridization showed less frequent heterogeneity compared with immunohistochemistry. Wherever possible, ERBB2 immunohistochemistry testing should be

  12. Evaluation of cytokeratin-19 in breast cancer tissue samples: a comparison of automatic and manual evaluations of scanned tissue microarray cylinders

    PubMed Central

    2015-01-01

    Background Digital image (DI) analysis avoids visual subjectivity in interpreting immunohistochemical stains and provides more reproducible results. An automated procedure consisting of two variant methods for quantifying the cytokeratin-19 (CK19) marker in breast cancer tissues is presented. Methods The first method (A) excludes the holes inside selected CK19 stained areas, and the second (B) includes them. 93 DIs scanned from complete cylinders of tissue microarrays were evaluated visually by two pathologists and by the automated procedures. Results and conclusions There was good concordance between the two automated methods, both of which tended to identify a smaller CK19-positive area than did the pathologists. The results obtained with method B were more similar to those of the pathologists; probably because it takes into account the entire positive tumoural area, including the holes. However, the pathologists overestimated the positive area of CK19. Further studies are needed to confirm the utility of this automated procedure in prognostic studies. PMID:26329009

  13. The manufacture and assessment of tissue microarrays: suggestions and criteria for analysis, with breast cancer as an example.

    PubMed

    Pinder, Sarah E; Brown, John P; Gillett, Cheryl; Purdie, Colin A; Speirs, Valerie; Thompson, Alastair M; Shaaban, Abeer M

    2013-03-01

    Tissue microarray (TMA) is an established and valuable tool, particularly in translational research and clinical trials, allowing resource-efficient use, and high-throughput profiling, of large numbers of tumours. Despite this, there is little evidence, or guidance, on the optimum manufacture, use and assessment of TMAs. Here we review some of the literature, using breast cancer as an example, to highlight good practice and pitfalls in the design and manufacture of TMAs. Issues, such as the size, number, spacing and layout of cores, as well as the assessment and reporting of studies using TMAs are addressed. We make some suggestions regarding these challenges, and propose a checklist of features that should be considered in order to stimulate debate and improve the quality of data produced by TMA analysis. PMID:23087330

  14. Analysis of differentially expressed genes in placental tissues of preeclampsia patients using microarray combined with the Connectivity Map database.

    PubMed

    Song, Y; Liu, J; Huang, S; Zhang, L

    2013-12-01

    Preeclampsia (PE), which affects 2-7% of human pregnancies, causes significant maternal and neonatal morbidity and mortality. To better understand the pathophysiology of PE, the gene expression profiles of placental tissue from 5 controls and 5 PE patients were assessed using microarray. A total of 224 transcripts were significantly differentially expressed (>2-fold change and q value <0.05, SAM software). Gene Ontology (GO) enrichment analysis indicated that genes involved in hypoxia and oxidative and reductive processes were significantly changed. Three differentially expressed genes (DEGs) involved in these biological processes were further verified by quantitative real-time PCR. Finally, the potential therapeutic agents for PE were explored via the Connectivity Map database. In conclusion, the data obtained in this study might provide clues to better understand the pathophysiology of PE and to identify potential therapeutic agents for PE patients.

  15. Role of β-catenin expression in paediatric mesenchymal lesions: a tissue microarray-based immunohistochemical study

    PubMed Central

    Santoro, A.; Pannone, G.; Errico, M.E.; Bifano, D.; Lastilla, G.; Bufo, P.; Loreto, C.; Donofrio, V.

    2012-01-01

    Beta-catenin is a major protein in the Wnt signalling pathway. Although it has been studied in various types of carcinoma, little is known about its expression in mesenchymal tumours. In this study 41 specimens of a variety of mesenchymal childhood tumours were compared to 24 samples of the corresponding adult tumours to assess the diagnostic value of nuclear β-catenin expression using tissue microarray-based immunohistochemistry. Similar to adult sarcoma and fibromatosis, β-catenin was not expressed in the majority of childhood sarcomas, and its nuclear translocation was detected in paediatric fibromatosis; non-negligible levels of nuclear staining in other tumour types demonstrate Wnt pathway activation in mesenchymal neoplasms of childhood and adolescence. PMID:23027341

  16. Re-Punching Tissue Microarrays Is Possible: Why Can This Be Useful and How to Do It

    PubMed Central

    Lacombe, Aurélien; Carafa, Vincenza; Schneider, Sandra; Sticker-Jantscheff, Melanie; Tornillo, Luigi; Eppenberger-Castori, Serenella

    2015-01-01

    Tissue microarray (TMA) methodology allows the concomitant analysis of hundreds of tissue specimens arrayed in the same manner on a recipient block. Subsequently, all samples can be processed under identical conditions, such as antigen retrieval procedure, reagent concentrations, incubation times with antibodies/probes, and escaping the inter-assays variability. Therefore, the use of TMA has revolutionized histopathology translational research projects and has become a tool very often used for putative biomarker investigations. TMAs are particularly relevant for large scale analysis of a defined disease entity. In the course of these exploratory studies, rare subpopulations can be discovered or identified. This can refer to subsets of patients with more particular phenotypic or genotypic disease with low incidence or to patients receiving a particular treatment. Such rare cohorts should be collected for more specific investigations at a later time, when, possibly, more samples of a rare identity will be available as well as more knowledge derived from concomitant, e.g., genetic, investigations will have been acquired. In this article we analyze for the first time the limits and opportunities to construct new TMA blocks using tissues from older available arrays and supplementary donor blocks. In summary, we describe the reasons and technical details for the construction of rare disease entities arrays.

  17. Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays

    PubMed Central

    Mutter, George L; Zahrieh, David; Liu, Chunmei; Neuberg, Donna; Finkelstein, David; Baker, Heather E; Warrington, Janet A

    2004-01-01

    Background Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted. Results 14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance. Conclusions Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment. PMID:15537428

  18. Current progress in 3D printing for cardiovascular tissue engineering.

    PubMed

    Mosadegh, Bobak; Xiong, Guanglei; Dunham, Simon; Min, James K

    2015-03-16

    3D printing is a technology that allows the fabrication of structures with arbitrary geometries and heterogeneous material properties. The application of this technology to biological structures that match the complexity of native tissue is of great interest to researchers. This mini-review highlights the current progress of 3D printing for fabricating artificial tissues of the cardiovascular system, specifically the myocardium, heart valves, and coronary arteries. In addition, how 3D printed sensors and actuators can play a role in tissue engineering is discussed. To date, all the work with building 3D cardiac tissues have been proof-of-principle demonstrations, and in most cases, yielded products less effective than other traditional tissue engineering strategies. However, this technology is in its infancy and therefore there is much promise that through collaboration between biologists, engineers and material scientists, 3D bioprinting can make a significant impact on the field of cardiovascular tissue engineering.

  19. Tissue stiffness dictates development, homeostasis, and disease progression.

    PubMed

    Handorf, Andrew M; Zhou, Yaxian; Halanski, Matthew A; Li, Wan-Ju

    2015-01-01

    Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression.

  20. Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

    PubMed Central

    Handorf, Andrew M; Zhou, Yaxian; Halanski, Matthew A; Li, Wan-Ju

    2015-01-01

    Abstract Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression. PMID:25915734

  1. Identification of Genes Associated With Progression and Metastasis of Advanced Cervical Cancers After Radiotherapy by cDNA Microarray Analysis

    SciTech Connect

    Harima, Yoko; Ikeda, Koshi; Utsunomiya, Keita; Shiga, Toshiko; Komemushi, Atsushi; Kojima, Hiroyuki; Nomura, Motoo; Kamata, Minoru; Sawada, Satoshi

    2009-11-15

    Purpose: To identify a set of genes related to the progression and metastasis of advanced cervical cancer after radiotherapy and to establish a predictive method. Methods and Materials: A total of 28 patients with cervical cancer (15 stage IIIB, 13 stage IVA patients) who underwent definitive radiotherapy between May 1995 and April 2001 were included in this study. All patients were positive for human papillomavirus infection and harbored the wild-type p53 gene. The expression profiles of 14 tumors with local failure and multiple distant metastasis and 14 tumors without metastasis (cancer free) obtained by punch biopsy were compared before treatment, using a cDNA microarray consisting of 23,040 human genes. Results: Sixty-three genes were selected on the basis of a clustering analysis, and the validity of these genes was confirmed using a cross-validation test. The most accurate prediction was achieved for 63 genes (sensitivity, 78.8%; specificity, 38.1%). Some of these genes were already known to be associated with metastasis via chromosomal instability (TTK, BUB1B), extracellular matrix components (matrix metalloproteinase 1 [MMP-1]), and carcinogenesis (protein phosphatase 1 regulatory subunit 7 [PPP1R7]). A 'predictive score' system was developed that could predict the probability for development of metastases using leave-one-out cross-validation methods. Conclusions: The present results may provide valuable information for identified predictive markers and novel therapeutic target molecules for progression and metastasis of advanced cervical cancer.

  2. Validation of a low-cost modified technique for constructing tissue microarrays for canine mammary tumor analysis.

    PubMed

    Silva, Franciele Basso Fernandes; Leite, Juliana da Silva; de Mello, Marcela Freire Vallim; Ferreira, Ana Maria Reis

    2016-09-01

    Compared with conventional histological paraffin blocks, tissue microarray (TMA) represents a "high-throughput tool" that provides rapid results, a time- and cost-effective approach and simultaneous investigation of several tissue samples under the same conditions. Given the large number of cases of dogs affected with mammary tumors, the complexity of these tumors and their similarity with breast cancer in women, this study aimed to validate a low-cost modified method to construct TMAs for canine mammary tumor analysis using immunomarkers. Carcinoma cases were selected from canine mammary carcinomas in mixed tumors (CMT) because this tumor type is the most heterogeneous among the histopathological types of mammary tumors observed in female dogs. Through a histopathological examination, tumor representativity was compared between conventional sections and histological sections obtained from the TMA block; both were stained with hematoxylin and eosin. An immunohistochemistry analysis was performed to compare the percentages of immunoreactive cells obtained in whole tissue sections versus those obtained from sections from the TMA block. Streptavidin-biotin peroxidase complex and anti-PCNA, anti-vimentin and anti-pancytokeratin antibodies were used. Statistical analysis consisted of the nonparametric Friedman's test (p≤0.05) and descriptive statistical analysis. Histopathological analysis showed tumor representativity in all TMA cores selected for the study. There was no difference between the immunohistochemical analysis of mammary tumors using conventional histological sections or sections obtained from a single 1-mm-diameter TMA core, regardless of the marker used: PCNA (p=0.279), pancytokeratin (p=0.243) and vimentin (p=0.967). The results did not change even when the means of any number of cores were compared among each other and with the conventional histological section: PCNA (p=0.413), pancytokeratin (p=0.177) and vimentin (p=1.0). Therefore, this study

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

    SciTech Connect

    Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith; Yin, John Liu; Byers, Richard

    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 in 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.

  4. 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. PMID:17570735

  5. Microarray gene expression analysis of fixed archival tissue permits molecular classification and identification of potential therapeutic targets in diffuse large B-cell lymphoma.

    PubMed

    Linton, Kim; Howarth, Christopher; Wappett, Mark; Newton, Gillian; Lachel, Cynthia; Iqbal, Javeed; Pepper, Stuart; Byers, Richard; Chan, Wing John; Radford, John

    2012-01-01

    Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) has a poor prognosis. Novel drugs targeting the constitutively activated NF-κB pathway characteristic of ABC-DLBCL are promising, but evaluation depends on accurate activated B cell-like (ABC)/germinal center B cell-like (GCB) molecular classification. This is traditionally performed on gene microarray expression profiles of fresh biopsies, which are not routinely collected, or by immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue, which lacks reproducibility and classification accuracy. We explored the possibility of using routine archival FFPE tissue for gene microarray applications. We examined Affymetrix HG U133 Plus 2.0 gene expression profiles from paired archival FFPE and fresh-frozen tissues of 40 ABC/GCB-classified DLBCL cases to compare classification accuracy and test the potential for this approach to aid the discovery of therapeutic targets and disease classifiers in DLBCL. Unsupervised hierarchical clustering of unselected present probe sets distinguished ABC/GCB in FFPE with remarkable accuracy, and a Bayesian classifier correctly assigned 32 of 36 cases with >90% probability. Enrichment for NF-κB genes was appropriately seen in ABC-DLBCL FFPE tissues. The top discriminatory genes expressed in FFPE separated cases with high statistical significance and contained novel biology with potential therapeutic insights, warranting further investigation. These results support a growing understanding that archival FFPE tissues can be used in microarray experiments aimed at molecular classification, prognostic biomarker discovery, and molecular exploration of rare diseases.

  6. Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer: A cross-sectional study.

    PubMed

    Oshiro, Hisashi; Czerniak, Bogdan A; Sakamaki, Kentaro; Tsuta, Koji; Bondaruk, Jolanta; Keyhani, Afsaneh; Dinney, Colin P; Nagai, Takeshi; Kamat, Ashish M

    2016-08-01

    Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples.Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland-Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers.Although the Bland-Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs.Our study suggests that the measurement results for certain biomarkers of bladder urothelial

  7. Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer

    PubMed Central

    Oshiro, Hisashi; Czerniak, Bogdan A.; Sakamaki, Kentaro; Tsuta, Koji; Bondaruk, Jolanta; Keyhani, Afsaneh; Dinney, Colin P.; Nagai, Takeshi; Kamat, Ashish M.

    2016-01-01

    Abstract Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples. Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland–Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers. Although the Bland–Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs. Our study suggests that the measurement results for certain biomarkers of bladder

  8. CD147 and Ki-67 overexpression confers poor prognosis in squamous cell carcinoma of oral tongue: A tissue microarray study

    PubMed Central

    Yu, Yau-Hua; Morales, Jose; Feng, Lei; Lee, J. Jack; El-Naggar, Adel K.; Vigneswaran, Nadarajah

    2015-01-01

    Squamous cell carcinoma of the oral tongue (SCCOT) exhibits high risk for recurrence and regional metastasis even after surgical resection. We assessed the clinicopathologic and prognostic significance of a group of functionally related biomarkers. We used a tissue microarray consisting SCCOT from 32 patients for this study. These patients were treated at the UT- M.D. Anderson Cancer Center from 1995 to 2008. Biomarker expression levels were examined by immunohistochemistry and graded semiquantitatively to determine their prognostic significance. CD147 and Tp63 expressions were significantly associated with a higher T-stage and Ki-67 labelling index as well as shorter overall survival (OS). Expression of Tp63 associated positively with poorly-differentiated histology. There was significant association of Tp63 with the expression levels of CD147 and Glut-1. Glut-1 overexpression was marginally associated with a higher T-stage. There was no prognostic significance of CD44v6 expression in SCCOT. SCCOT with CD147 overexpression in combination with high Ki-67 labelling index had poor OS. CD147 and Ki-67 overexpression is associated with aggressive disease with poor prognosis in SCCOT. PMID:25747176

  9. Targeting topoisomerase IIa in endometrial adenocarcinoma: a combined chromogenic in situ hybridization and immunohistochemistry study based on tissue microarrays.

    PubMed

    Tsiambas, E; Alexopoulou, D; Lambropoulou, S; Gerontopoulos, K; Karakitsos, P; Karameris, A

    2006-01-01

    Topoisomerase IIa is a nucleic enzyme that affects the topological structure of DNA and also is a target for chemotherapy (ie, anthracyclines). In this study, we coevaluated its protein expression with chromosome 17 and gene status. Using tissue microarrays, 40 cases of sporadic, primary endometrial adenocarcinomas, 5 cases of atypical hyperplasia, and 5 cases of benign hyperplasia were obtained and reembedded into two paraffin blocks with a core diameter of 1 mm. Immunohistochemistry combined with chromogenic in situ hybridization was performed in 2 and 5 microm sections, respectively. Finally using a semiautomated Image Analysis System, we evaluated the levels of Nuclear labeling index of topoisomerase IIa expression. Statistical analysis was performed by SPSS version 11.0 software. The results indicate that chromosome 17 instability (aneuploidy in 7/40 cases) and Topo IIa gene deregulation (amplification in 3/40 and deletion in 1/40 cases) are significant genetic events correlated with biologic behavior in endometrial adenocarcinoma. Because protein overexpression was observed in a significant proportion of the tumors (18/40), detection of the specific gene deregulation mechanism is a crucial process for application of targeted chemotherapies, which are characterized by different levels of cardiotoxicity and other serious effects.

  10. Detection of Epstein-Barr virus in children and adolescents with Burkitt's lymphoma by in situ hybridization using tissue microarrays.

    PubMed

    Pizza, Maria; Bruniera, Paula; Luporini, Silvia Maria; Marcelino da Silva, Helena Reis; Borsato, Maria Luisa; de Castro, Helaine Cristina; Soares, Fernando Augusto; Paes, Roberto Antoônio Pinto

    2008-04-01

    In this retrospective study, we evaluated 53 patients diagnosed with Burkitt's lymphoma (BL). Patients were admitted to the Pediatric Department of Santa Casa de São Paulo, Brazil from August 1988 to September 2006. The male gender was found more frequently in the proportion of three males to one female. At the time of the diagnosis, the mean age was 6 years. The most frequent clinical presentation was abdominal (extra nodal) in 42 (79.2%) cases. The patients were treated with chemotherapy using two different treatment methods, 30 cases until September 1994 with BGLTI -87 and the other 23 until September of 2006 with BGLTI -96. According to Murphy's classification, 90.2% of the patients were diagnosed as stages III and VI showing an advanced disease at time of diagnosis. Using Tissue Microarrays (TMA); we searched for the expression of the Epstein-Barr virus (EBV) RNA in 50 patients. The presence of EBV using in situ hybridization was found in 33/50 (66%) and there was no association between the presence of the virus in the tumor cells or patient age, as well as the survival rate. There was 74.5% global survival rate until September, 2005 of 12 years and 8 months. The disease free survival rate was 86.5%. The frequency of the EBV association with this neoplasia in Brazil is intermediate between the endemic and sporadic forms of disease.

  11. Classification of MALDI-MS imaging data of tissue microarrays using canonical correlation analysis-based variable selection.

    PubMed

    Winderbaum, Lyron; Koch, Inge; Mittal, Parul; Hoffmann, Peter

    2016-06-01

    Applying MALDI-MS imaging to tissue microarrays (TMAs) provides access to proteomics data from large cohorts of patients in a cost- and time-efficient way, and opens the potential for applying this technology in clinical diagnosis. The complexity of these TMA data-high-dimensional low sample size-provides challenges for the statistical analysis, as classical methods typically require a nonsingular covariance matrix that cannot be satisfied if the dimension is greater than the sample size. We use TMAs to collect data from endometrial primary carcinomas from 43 patients. Each patient has a lymph node metastasis (LNM) status of positive or negative, which we predict on the basis of the MALDI-MS imaging TMA data. We propose a variable selection approach based on canonical correlation analysis that explicitly uses the LNM information. We apply LDA to the selected variables only. Our method misclassifies 2.3-20.9% of patients by leave-one-out cross-validation and strongly outperforms LDA after reduction of the original data with principle component analysis. PMID:27028088

  12. Severe progressive periodontal destruction due to radiation tissue injury.

    PubMed

    Yusof, Z W; Bakri, M M

    1993-12-01

    Cancer radiotherapy to the head and neck region results in short- and long-term radiation tissue injuries. Radiation bone injury is a long-term manifestation which could progress to osteoradionecrosis. A case of radiation tissue injury to the periodontium is presented. The possible pathogenesis of these events is described as they relate to the sequential radiographic changes observed over a period of 6 years until the involved teeth were exfoliated. The post-irradiation management of the teeth with advancing periodontal disease in the path of irradiation was by conservative means, including good personal oral hygiene care, scaling and root planing, periodic chlorhexidine irrigation, and topical fluoride application.

  13. Use of principal components analysis and protein microarray to explore the association of HIV-1-specific IgG responses with disease progression.

    PubMed

    Gerns Storey, Helen L; Richardson, Barbra A; Singa, Benson; Naulikha, Jackie; Prindle, Vivian C; Diaz-Ochoa, Vladimir E; Felgner, Phil L; Camerini, David; Horton, Helen; John-Stewart, Grace; Walson, Judd L

    2014-01-01

    The role of HIV-1-specific antibody responses in HIV disease progression is complex and would benefit from analysis techniques that examine clusterings of responses. Protein microarray platforms facilitate the simultaneous evaluation of numerous protein-specific antibody responses, though excessive data are cumbersome in analyses. Principal components analysis (PCA) reduces data dimensionality by generating fewer composite variables that maximally account for variance in a dataset. To identify clusters of antibody responses involved in disease control, we investigated the association of HIV-1-specific antibody responses by protein microarray, and assessed their association with disease progression using PCA in a nested cohort design. Associations observed among collections of antibody responses paralleled protein-specific responses. At baseline, greater antibody responses to the transmembrane glycoprotein (TM) and reverse transcriptase (RT) were associated with higher viral loads, while responses to the surface glycoprotein (SU), capsid (CA), matrix (MA), and integrase (IN) proteins were associated with lower viral loads. Over 12 months greater antibody responses were associated with smaller decreases in CD4 count (CA, MA, IN), and reduced likelihood of disease progression (CA, IN). PCA and protein microarray analyses highlighted a collection of HIV-specific antibody responses that together were associated with reduced disease progression, and may not have been identified by examining individual antibody responses. This technique may be useful to explore multifaceted host-disease interactions, such as HIV coinfections.

  14. Nerve Fibers in Breast Cancer Tissues Indicate Aggressive Tumor Progression

    PubMed Central

    Huang, Di; Su, Shicheng; Cui, Xiuying; Shen, Ximing; Zeng, Yunjie; Wu, Wei; Chen, Jianing; Chen, Fei; He, Chonghua; Liu, Jiang; Huang, Wei; Liu, Qiang; Su, Fengxi; Song, Erwei; Ouyang, Nengtai

    2014-01-01

    Abstract Emerging evidence has indicated nerve fibers as a marker in the progression of various types of cancers, such as pancreatic cancer and prostate cancer. However, whether nerve fibers are associated with breast cancer progression remains unclear. In this study, we evaluated the presence of nerve fibers in 352 breast cancer specimens and 83 benign breast tissue specimens including 43 cases of cystic fibrosis and 40 cases of fibroadenoma from 2 independent breast tumor center using immunohistochemical staining for specific peripheral nerve fiber markers. In all, nerve fibers were present in 130 out of 352 breast cancer tissue specimens, while none were detected in normal breast tissue specimens. Among 352 cases, we defined 239 cases from Sun Yat-Sen Memorial Hospital, Guangzhou, China, as the training set, and 113 cases from the First Affiliated Hospital of Shantou University, Guangdong, China, as the validation set. The thickness of tumor-involving nerve fibers is significantly correlated with poor differentiation, lymph node metastasis, high clinical staging, and triple negative subtype in breast cancer. More importantly, Cox multifactor analysis indicates that the thickness of tumor-involving nerve fibers is a previously unappreciated independent prognostic factors associated with shorter disease-free survival of breast cancer patients. Our findings are further validated by online Oncomine database. In conclusion, our results show that nerve fiber involvement in breast cancer is associated with progression of the malignancy and warrant further studies in the future. PMID:25501061

  15. Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

    PubMed

    Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

    2015-12-01

    Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration.

  16. Dephosphorylated cofilin expression is associated with poor prognosis in cases of human breast cancer: a tissue microarray analysis

    PubMed Central

    Maimaiti, Yusufu; Liu, Zeming; Tan, Jie; Abudureyimu, Kelimu; Huang, Bangxing; Liu, Chunping; Guo, Yawen; Wang, Changwen; Nie, Xiu; Zhou, Jing; Huang, Tao

    2016-01-01

    Background Proteins in the cofilin pathway regulate actin dynamics and may be involved in cancer cell migration and invasion. However, there are no direct data that suggest that dephosphorylated cofilin can affect breast cancer prognosis. Methods We assessed the expressions of cofilin and phosphorylated cofilin (P-cofilin) in breast cancer tissue microarrays (290 patients, mean follow-up: 95.7±2.49 months) to evaluate dephosphorylated cofilin and its relationship with breast cancer prognosis. The associations of pathological characteristics with cumulative survival were evaluated using Kaplan–Meier analysis. Results Univariate analyses revealed that overall survival was associated with cofilin levels, N category, TNM stage, estrogen receptor status, progesterone receptor status, and molecular subtypes. Cofilin status and TNM stage independently affected overall survival, although P-cofilin expression was not associated with patient survival. In the P-cofilin-negative subgroup, cofilin expression was significantly associated with patient survival, although cofilin expression was not significantly associated with patient survival in the P-cofilin-positive subgroup. We further analyzed the P-cofilin-negative cases and found that Ki-67 expression was significantly elevated in the subgroup that was strongly positive for cofilin (P=0.002). Conclusion Among P-cofilin-negative patients with breast cancer, cofilin expression defines a population of patients with lower overall survival, which suggests that dephosphorylated cofilin expression might predict the prognosis in cases of P-cofilin-negative breast cancer. Furthermore, our results suggest that inhibitors of dephosphorylated cofilin expression may provide therapeutic benefits in patients with breast cancer. PMID:27799793

  17. Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

    PubMed

    Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

    2015-12-01

    Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. PMID:26092703

  18. Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma. A tissue microarray study of 158 cases.

    PubMed

    Compérat, Eva; Camparo, Philippe; Haus, Rachel; Chartier-Kastler, Emmanuel; Bart, Stephane; Delcourt, Annick; Houlgatte, Alain; François, Richard; Capron, Fréderique; Vieillefond, Annick

    2006-03-01

    P63 is a member of the p53 family, which plays a role in the differentiation of urothelium and is supposed to play a role in urothelial carcinogenesis. P53 and MIB-1 are recognised in many studies as predictive markers of progression, but few studies in the literature have examined p63. The aims of our study were to explore the expression of p63 in bladder carcinomas and to compare this expression to p53 and MIB-1, as well as to stage and grade. Tissue microarrays were performed on 158 urothelial carcinomas (56 pTa, 45 pT1 and 57>or=pT2). Immunohistochemical studies were performed with p63, p53 and MIB-1 antibodies. In our study we observed that p63 immunostaining is present in all cell layers in papillary urothelial neoplasm of low malignant potential (PUNLMP), but partially lost in non-invasive papillary urothelial carcinoma low grade (NILGC) and in pT1/>or=pT2 bladder cancers. P53 and MIB-1 displayed lower expression in PUNLMP/NILGC vs non-invasive papillary urothelial carcinoma high grade (NIHGC)/pT1, but there was no correlation between the expression of p63, p53 and MIB-1. Our study demonstrates that p63 expression distinguishes between PUNLMP/NILGC and NIHGC/pT1 (p=4.10(5)). A statistical difference disserving pTa and pT1/>or=pT2 with a statistical significance (p<10(-6)) could also be observed. P63 should be considered as an additional biomarker that might help pathologists to classify their patients.

  19. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

    PubMed

    Chen, Xi; Deane, Natasha G; Lewis, Keeli B; Li, Jiang; Zhu, Jing; Washington, M Kay; Beauchamp, R Daniel

    2016-01-01

    The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections. PMID:27176004

  20. [Progress of Autoantibody Examinations for Connective Tissue Diseases].

    PubMed

    Akashi, Kengo; Saegusa, Jun; Morinobu, Akio

    2015-05-01

    Connective tissue diseases are chronic inflammatory diseases that can affect multiple organs and, thus, have a broad spectrum of clinical presentations. Various autoantibodies are detected in patients with connective tissue diseases, represented by anti-nuclear antibody for systemic lupus erythematosus (SLE), systemic sclerosis (SSc), polymyositis/dermatomyositis (PM/DM), Sjögren's syndrome, and mixed connective tissue disease. Assessment of the autoantibody profile is fundamental for the clinical management of patients with connective tissue diseases, providing important data for the diagnosis, clinical characterization, and disease activity evaluation. Anti-ribosomal P antibody and anti-NMDA receptor antibody are associated with neuropsychiatric SLE. Anti-synthetase syndrome comprises the association of myositis (PM/DM), interstitial lung disease, fever, Raynaud's phenomenon, mechanic's hands, and anti-aminoacyl tRNA synthetase antibodies. Anti-MDA5 antibody is detected in patients with clinically amyopathic DM, often complicated by rapidly progressive interstitial lung disease. Between 50 and 75% of malignancy-associated DM patients are positive for anti-TIF1-γ antibody. Anti-RNA polymerase III antibody is associated with diffuse cutaneous SSc and renal crisis. This review focuses on the importance and usefulness of these autoantibodies for the diagnosis and management of patients with connective tissue diseases in clinical practice.

  1. Microhardness of human cancellous bone tissue in progressive hip osteoarthritis.

    PubMed

    Tomanik, Magdalena; Nikodem, Anna; Filipiak, Jarosław

    2016-12-01

    Bone tissue is a biological system in which the dynamic processes of, among others, bone formation or internal reconstruction will determine the spatial structure of the tissue and its mechanical properties. The appearance of a factor disturbing the balance between biological processes, e.g. a disease, will cause changes in the spatial structure of bones, thus affecting its mechanical properties. One of the bone diseases most common in an increasingly ageing population is osteoarthritis, also referred to as degenerative joint disease. It is estimated that in 2050 about 1300 million people will show symptoms of OA. The appearance of a pathological stimulus disturbs the balance of the processes of degradation and synthesis of articular cartilage, chondrocytes and the extracellular matrix, and the subchondral bone layer. As osteoarthritis progresses, study of the epiphysis reveals increasingly widespread changes of the articular surface and the internal structure of bone tissue. In this paper, the authors point out the differences in the mechanical properties of cancellous bone tissue forming the proximal epiphysis of the femoral bone during the progressive stages of OA. In order to determine microproperties of bone trabeculae, specimens from different stages of the disease (N=9) were subjected to microindentation testing, which made it possible to determine the material properties of bone tissue, such as microhardness HV and Young׳s modulus E. In addition, mechanical tests were supplemented with Raman spectroscopy, which determine the degree of bone mineralization, and measurements of structural properties based on analysis using microCT. The conducted tests were used to establish both quantitative and quantitative description of changes in the structural and mechanical properties connected with reorganization of trabeculae making up the bone in the various stages of osteoarthritis. The proposed description will supplement existing knowledge in the literature about

  2. Microhardness of human cancellous bone tissue in progressive hip osteoarthritis.

    PubMed

    Tomanik, Magdalena; Nikodem, Anna; Filipiak, Jarosław

    2016-12-01

    Bone tissue is a biological system in which the dynamic processes of, among others, bone formation or internal reconstruction will determine the spatial structure of the tissue and its mechanical properties. The appearance of a factor disturbing the balance between biological processes, e.g. a disease, will cause changes in the spatial structure of bones, thus affecting its mechanical properties. One of the bone diseases most common in an increasingly ageing population is osteoarthritis, also referred to as degenerative joint disease. It is estimated that in 2050 about 1300 million people will show symptoms of OA. The appearance of a pathological stimulus disturbs the balance of the processes of degradation and synthesis of articular cartilage, chondrocytes and the extracellular matrix, and the subchondral bone layer. As osteoarthritis progresses, study of the epiphysis reveals increasingly widespread changes of the articular surface and the internal structure of bone tissue. In this paper, the authors point out the differences in the mechanical properties of cancellous bone tissue forming the proximal epiphysis of the femoral bone during the progressive stages of OA. In order to determine microproperties of bone trabeculae, specimens from different stages of the disease (N=9) were subjected to microindentation testing, which made it possible to determine the material properties of bone tissue, such as microhardness HV and Young׳s modulus E. In addition, mechanical tests were supplemented with Raman spectroscopy, which determine the degree of bone mineralization, and measurements of structural properties based on analysis using microCT. The conducted tests were used to establish both quantitative and quantitative description of changes in the structural and mechanical properties connected with reorganization of trabeculae making up the bone in the various stages of osteoarthritis. The proposed description will supplement existing knowledge in the literature about

  3. HER2 in gastric cancer: an immunohistochemical study on tissue microarrays and the corresponding whole-tissue sections with a supplemental fish study.

    PubMed

    Gasljevic, Gorana; Lamovec, Janez; Contreras, Juan Antonio; Zadnik, Vesna; Blas, Mateja; Gasparov, Slavko

    2013-10-01

    Since focal HER2 expression is an issue in GC, TMA construction from the paraffin-embedded surgically-obtained tissue may not reflect its real status. The aim of this study was to assess the HER2 status in tissue microarrays (TMAs) and the corresponding whole sections using HercepTest immunohistochemistry (IHC), and to correlate it and to assess the concordance of HER2 IHC and fluorescence in situ hybridization (FISH) in TMAs. Concordance of the HER2 expression status for 302 cases of gastric cancer using 9 paired TMAs was evaluated using a 2-mm core size and 305 corresponding whole sections. Concordance of the IHC and FISH HER2 status was compared. In addition,, the HER2 status was compared to clinicopathological characteristics and patients' survival. Using the whole-section approach, HER2 over-expression was found in 25.2 % (HER2 3+ 6.6 %, HER2 2+ 18.7 %) of tumours. The overall concordance of IHC between the cores and the whole section was 84.9 %; 15.1 % of the tumours showed HER2 amplification. The overall concordance of IHC and FISH on cores was 75.7 %. The level of amplification correlated with the IHC score. Relationship between the intestinal and papillary types and tumour grade was observed for tumours with over-expression and amplification, whereas tumour location was related only to over-expression. There was a statistically significant difference in the overall survival of the patients, which was related to HER2 amplification. In conclusion, good concordance of the IHC HER2 results between tissue cores in TMA and whole sections, and excellent concordance of the IHC and FISH results on tissue cores was found. At least a part of the observed IHC HER2 heterogeneity could very likely be explained by fixation artifacts. With adequate fixation, a higher concordance of IHC HER2 between the cores and the whole sections can be expected. The TMA approach could enable an easier analysis of more than one representative tumour block.

  4. Interlaboratory Performance of a Microarray-Based Gene Expression Test to Determine Tissue of Origin in Poorly Differentiated and Undifferentiated Cancers

    PubMed Central

    Dumur, Catherine I.; Lyons-Weiler, Maureen; Sciulli, Christin; Garrett, Carleton T.; Schrijver, Iris; Holley, Tara K.; Rodriguez-Paris, Juan; Pollack, Jonathan R.; Zehnder, James L.; Price, Melissa; Hagenkord, Jill M.; Rigl, C. Ted; Buturovic, Ljubomir J.; Anderson, Glenda G.; Monzon, Federico A.

    2008-01-01

    Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48 ± 3.97) and κ statistics (κ > 0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories. PMID:18083688

  5. [Metastasis and progression mechanisms of soft tissue tumors].

    PubMed

    Steinestel, K; Wardelmann, E

    2015-11-01

    Invasion and metastatic dissemination of tumor cells defines prognosis not only in patients with epithelial, but also mesenchymal neoplasms. Early and clinically inapparent micrometastases occur in many patients, and the risk for metastasis correlates with the tumor subtype and histologic tumor grade. In recent years and analogous to the situation in epithelial tumors, mechanisms of tumor cell dissemination in soft tissue tumors have been increasingly understood, and it has been shown that reorganization of the actin cytoskeleton plays a key role in these processes. This review summarizes current knowledge on the mechanisms of progression and metastasis of soft tissue tumors and points out possible targets for novel anti-invasive and anti-metastatic therapies. PMID:26324521

  6. High‐throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium

    PubMed Central

    Howat, William J; Daley, Frances; Zabaglo, Lila; McDuffus, Leigh‐Anne; Blows, Fiona; Coulson, Penny; Raza Ali, H; Benitez, Javier; Milne, Roger; Brenner, Herman; Stegmaier, Christa; Mannermaa, Arto; Chang‐Claude, Jenny; Rudolph, Anja; Sinn, Peter; Couch, Fergus J; Tollenaar, Rob A.E.M.; Devilee, Peter; Figueroa, Jonine; Sherman, Mark E; Lissowska, Jolanta; Hewitt, Stephen; Eccles, Diana; Hooning, Maartje J; Hollestelle, Antoinette; WM Martens, John; HM van Deurzen, Carolien; Investigators, kConFab; Bolla, Manjeet K; Wang, Qin; Jones, Michael; Schoemaker, Minouk; Broeks, Annegien; van Leeuwen, Flora E; Van't Veer, Laura; Swerdlow, Anthony J; Orr, Nick; Dowsett, Mitch; Easton, Douglas; Schmidt, Marjanka K; Pharoah, Paul D; Garcia‐Closas, Montserrat

    2016-01-01

    Abstract Automated methods are needed to facilitate high‐throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large‐scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performance in studies from the Breast Cancer Association Consortium. We utilized 166 TMAs containing 16,953 tumour cores representing 9,059 breast cancer cases, from 13 studies, with information on other clinical and pathological characteristics. TMAs were stained for Ki67 using standard immunohistochemical procedures, and scanned and digitized using the Ariol system. An automated algorithm was developed for the scoring of Ki67, and scores were compared to computer assisted visual (CAV) scores in a subset of 15 TMAs in a training set. We also assessed the correlation between automated Ki67 scores and other clinical and pathological characteristics. Overall, we observed good discriminatory accuracy (AUC = 85%) and good agreement (kappa = 0.64) between the automated and CAV scoring methods in the training set. The performance of the automated method varied by TMA (kappa range= 0.37–0.87) and study (kappa range = 0.39–0.69). The automated method performed better in satisfactory cores (kappa = 0.68) than suboptimal (kappa = 0.51) cores (p‐value for comparison = 0.005); and among cores with higher total nuclei counted by the machine (4,000–4,500 cells: kappa = 0.78) than those with lower counts (50–500 cells: kappa = 0.41; p‐value = 0.010). Among the 9,059 cases in this study, the correlations between automated Ki67 and clinical and pathological characteristics were found to be in the expected directions. Our findings indicate that automated scoring of Ki67 can be an efficient method to obtain good quality data across large numbers of TMAs from multicentre studies. However, robust algorithm development and rigorous pre‐ and post

  7. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  8. Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in Drosophila

    PubMed Central

    Hackney, Jennifer F.; Zolali-Meybodi, Omid; Cherbas, Peter

    2012-01-01

    In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling. PMID:23166607

  9. Genome-wide effects of acute progressive feed restriction in liver and white adipose tissue

    SciTech Connect

    Pohjanvirta, Raimo Boutros, Paul C.; Moffat, Ivy D.; Linden, Jere; Wendelin, Dominique; Okey, Allan B.

    2008-07-01

    Acute progressive feed restriction (APFR) represents a specific form of caloric restriction in which feed availability is increasingly curtailed over a period of a few days to a few weeks. It is often used for control animals in toxicological and pharmacological studies on compounds causing body weight loss to equalize weight changes between experimental and control groups and thereby, intuitively, to also set their metabolic states to the same phase. However, scientific justification for this procedure is lacking. In the present study, we analyzed by microarrays the impact on hepatic gene expression in rats of two APFR regimens that caused identical diminution of body weight (19%) but differed slightly in duration (4 vs. 10 days). In addition, white adipose tissue (WAT) was also subjected to the transcriptomic analysis on day-4. The data revealed that the two regimens led to distinct patterns of differentially expressed genes in liver, albeit some major pathways of energy metabolism were similarly affected (particularly fatty acid and amino acid catabolism). The reason for the divergence appeared to be entrainment by the longer APFR protocol of peripheral oscillator genes, which resulted in derailment of circadian rhythms and consequent interaction of altered diurnal fluctuations with metabolic adjustments in gene expression activities. WAT proved to be highly unresponsive to the 4-day APFR as only 17 mRNA levels were influenced by the treatment. This study demonstrates that body weight is a poor proxy of metabolic state and that the customary protocols of feed restriction can lead to rhythm entrainment.

  10. Gene expression profiling of subcutaneous adipose tissue in morbid obesity using a focused microarray: Distinct expression of cell-cycle- and differentiation-related genes

    PubMed Central

    2010-01-01

    Background Obesity results from an imbalance between food intake and energy expenditure, which leads to an excess of adipose tissue. The excess of adipose tissue and adipocyte dysfunction associated with obesity are linked to the abnormal regulation of adipogenesis. The objective of this study was to analyze the expression profile of cell-cycle- and lipid-metabolism-related genes of adipose tissue in morbid obesity. Methods We used a custom-made focused cDNA microarray to determine the adipose tissue mRNA expression profile. Gene expression of subcutaneous abdominal fat samples from 15 morbidly obese women was compared with subcutaneous fat samples from 10 nonobese control patients. The findings were validated in an independent population of 31 obese women and 9 obese men and in an animal model of obesity (Lepob/ob mice) by real-time RT-PCR. Results Microarray analysis revealed that transcription factors that regulate the first stages of adipocyte differentiation, such as CCAAT/enhancer binding protein beta (C/EBPβ) and JUN, were upregulated in the adipose tissues of morbidly obese patients. The expression of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor which controls lipid metabolism and the final steps of preadipocyte conversion into mature adipocytes, was downregulated. The expression of three cyclin-dependent kinase inhibitors that regulate clonal expansion and postmitotic growth arrest during adipocyte differentiation was also altered in obese subjects: p18 and p27 were downregulated, and p21 was upregulated. Angiopoietin-like 4 (ANGPTL4), which regulates angiogenesis, lipid and glucose metabolism and it is know to increase dramatically in the early stages of adipocyte differentiation, was upregulated. The expression of C/EBPβ, p18, p21, JUN, and ANGPTL4 presented similar alterations in subcutaneous adipose tissue of Lepob/ob mice. Conclusions Our microarray gene profiling study revealed that the expression of genes

  11. Microarray Analysis in Glioblastomas

    PubMed Central

    Bhawe, Kaumudi M.; Aghi, Manish K.

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930–2942, 2012)To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013)To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59–70, 2013; Verhaak et al., Cancer Cell 17(1):98–110, 2010) While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  12. Microarray Analysis in Glioblastomas.

    PubMed

    Bhawe, Kaumudi M; Aghi, Manish K

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: i. To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930-2942, 2012). ii. To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013). iii. To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59-70, 2013; Verhaak et al., Cancer Cell 17(1):98-110, 2010). While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  13. Functional Protein Microarray Technology

    PubMed Central

    Hu, Shaohui; Xie, Zhi; Qian, Jiang; Blackshaw, Seth; Zhu, Heng

    2010-01-01

    Functional protein microarrays are emerging as a promising new tool for large-scale and high-throughput studies. In this article, we will review their applications in basic proteomics research, where various types of assays have been developed to probe binding activities to other biomolecules, such as proteins, DNA, RNA, small molecules, and glycans. We will also report recent progress of using functional protein microarrays in profiling protein posttranslational modifications, including phosphorylation, ubiquitylation, acetylation, and nitrosylation. Finally, we will discuss potential of functional protein microarrays in biomarker identification and clinical diagnostics. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology. PMID:20872749

  14. A predictive factor of the quality of microarray comparative genomic hybridization analysis for formalin-fixed paraffin-embedded archival tissue.

    PubMed

    Nakao, Kenjiro; Oikawa, Masahiro; Arai, Junichi; Mussazhanova, Zhanna; Kondo, Hisayoshi; Shichijo, Kazuko; Nakashima, Masahiro; Hayashi, Tomayoshi; Yoshiura, Koh-Ichiro; Hatachi, Toshiko; Nagayasu, Takeshi

    2013-09-01

    Utilizing formalin-fixed paraffin-embedded (FFPE) archival tissue, the most common form of tissue preservation in routine practice, for cytogenetic analysis using microarray comparative genomic hybridization (aCGH) remains challenging. We searched for a predictive factor of the performance of FFPE DNA in aCGH analysis. DNA was extracted from 63 FFPE archival tissue samples of various tissue types (31 breast cancers, 24 lung cancers, and 8 thyroid tumors), followed by aCGH analysis using high-density oligonucleotide microarrays. Tumor DNA from matched frozen samples and from FFPE samples after whole-genome amplification were also analyzed in 2 and 4 case, respectively. The derivative log ratio spread (DLRSpread) was used to assess the overall quality of each aCGH result. The DLRSpread correlated significantly with the double-stranded DNA ratio of tumor DNA, storage time, and the degree of labeling with Cy5 (P<0.0001; correlation coefficients=-0.796, 0.551, -0.481, respectively). Stepwise multiple linear regression analysis revealed that the double-stranded DNA ratio of tumor DNA is the most significant predictive factor of DLRSpread (regression coefficient=-0.4798; P=<0.0001). The cytogenetic profiles of FFPE and matched frozen samples showed good concordance. Although the double-stranded DNA ratios were increased after whole-genome amplification, the DLRSpread was not improved. The double-stranded DNA ratio can be used to predict the performance of aCGH analysis for DNA from FFPE samples. Using this quality metric, valuable FFPE archival tissue samples can be utilized for aCGH analysis.

  15. 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 Central

    HU, XIAOYU; YANG, ZHU; ZENG, MANMAN; LIU, YI; YANG, XIAOTAO; LI, YANAN; LI, XU; YU, QIUBO

    2016-01-01

    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. PMID:27347196

  16. 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…

  17. Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.

    PubMed

    Hovestad-Bijl, Liesbeth; van Ameijde, Jeroen; Pijnenburg, Dirk; Hilhorst, Riet; Liskamp, Rob; Ruijtenbeek, Rob

    2016-01-01

    A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases. PMID:27514800

  18. Rat Mitochondrion-Neuron Focused Microarray (rMNChip) and Bioinformatics Tools for Rapid Identification of Differential Pathways in Brain Tissues

    PubMed Central

    Su, Yan A.; Zhang, Qiuyang; Su, David M.; Tang, Michael X.

    2011-01-01

    Mitochondrial function is of particular importance in brain because of its high demand for energy (ATP) and efficient removal of reactive oxygen species (ROS). We developed rat mitochondrion-neuron focused microarray (rMNChip) and integrated bioinformatics tools for rapid identification of differential pathways in brain tissues. rMNChip contains 1,500 genes involved in mitochondrial functions, stress response, circadian rhythms and signal transduction. The bioinformatics tool includes an algorithm for computing of differentially expressed genes, and a database for straightforward and intuitive interpretation for microarray results. Our application of these tools to RNA samples derived from rat frontal cortex (FC), hippocampus (HC) and hypothalamus (HT) led to the identification of differentially-expressed signal-transduction-bioenergenesis and neurotransmitter-synthesis pathways with a dominant number of genes (FC/HC = 55/6; FC/HT = 55/4) having significantly (p<0.05, FDR<10.70%) higher (≥1.25 fold) RNA levels in the frontal cortex than the others, strongly suggesting active generation of ATP and neurotransmitters and efficient removal of ROS. Thus, these tools for rapid and efficient identification of differential pathways in brain regions will greatly facilitate our systems-biological study and understanding of molecular mechanisms underlying complex and multifactorial neurodegenerative diseases. PMID:21494430

  19. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  20. Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

    PubMed

    Horn, Heike; Bausinger, Julia; Staiger, Annette M; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

  1. [Progress on strategies to promote vascularization in bone tissue engineering].

    PubMed

    Chen, Kai; Zhang, Chao; Wang, Lu; Mao, Yu-Yan; Lu, Jian-Xi; Chen, Lei

    2015-04-01

    With the continuous development of bone tissue engineering, a variety of emerging bone graft materials provided various methods for repairing bone defects. Early and rapid accomplishment of revascularization of materials interior after implantation of bone transplantation materials is a difficulty faced to bone tissue engineering. Blood vessels ingrowth provides the requisite netritional support for the regeneration reconstruction of bone tissue, for this reason, vascularization plays a significant role in bone tissue engineering. However,there is not a golden standard strategy of vascularization at present. Scaffold materials, cells and growth factors still are three indispensable elements in tissue engineering, and are cardinal points of the promoting vascularization strategies. Multiple growth factors or multiple cells combined with scaffolds, which are hot spots, have obtained excellent vascularization. This review focused on the comprehensive strategies for promoting the successful vascularization of tissue engineered scaffolds.

  2. Hydrogel scaffolds for tissue engineering: Progress and challenges

    PubMed Central

    El-Sherbiny, Ibrahim M.; Yacoub, Magdi H.

    2013-01-01

    Designing of biologically active scaffolds with optimal characteristics is one of the key factors for successful tissue engineering. Recently, hydrogels have received a considerable interest as leading candidates for engineered tissue scaffolds due to their unique compositional and structural similarities to the natural extracellular matrix, in addition to their desirable framework for cellular proliferation and survival. More recently, the ability to control the shape, porosity, surface morphology, and size of hydrogel scaffolds has created new opportunities to overcome various challenges in tissue engineering such as vascularization, tissue architecture and simultaneous seeding of multiple cells. This review provides an overview of the different types of hydrogels, the approaches that can be used to fabricate hydrogel matrices with specific features and the recent applications of hydrogels in tissue engineering. Special attention was given to the various design considerations for an efficient hydrogel scaffold in tissue engineering. Also, the challenges associated with the use of hydrogel scaffolds were described. PMID:24689032

  3. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  4. Recent progress in interfacial tissue engineering approaches for osteochondral defects.

    PubMed

    Castro, Nathan J; Hacking, S Adam; Zhang, Lijie Grace

    2012-08-01

    This review provides a brief synopsis of the anatomy and physiology of the osteochondral interface, scaffold-based and non-scaffold based approaches for engineering both tissues independently as well as recent developments in the manufacture of gradient constructs. Novel manufacturing techniques and nanotechnology will be discussed with potential application in osteochondral interfacial tissue engineering. PMID:22677924

  5. Fiber-Based Tissue Engineering: Progress, Challenges, and Opportunities

    PubMed Central

    Tamayol, Ali; Akbari, Mohsen; Annabi, Nasim; Paul, Arghya; Khademhosseini, Ali; Juncker, David

    2013-01-01

    Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the above mentioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice. PMID:23195284

  6. Analyzing illumina gene expression microarray data from different tissues: methodological aspects of data analysis in the metaxpress consortium.

    PubMed

    Schurmann, Claudia; Heim, Katharina; Schillert, Arne; Blankenberg, Stefan; Carstensen, Maren; Dörr, Marcus; Endlich, Karlhans; Felix, Stephan B; Gieger, Christian; Grallert, Harald; Herder, Christian; Hoffmann, Wolfgang; Homuth, Georg; Illig, Thomas; Kruppa, Jochen; Meitinger, Thomas; Müller, Christian; Nauck, Matthias; Peters, Annette; Rettig, Rainer; Roden, Michael; Strauch, Konstantin; Völker, Uwe; Völzke, Henry; Wahl, Simone; Wallaschofski, Henri; Wild, Philipp S; Zeller, Tanja; Teumer, Alexander; Prokisch, Holger; Ziegler, Andreas

    2012-01-01

    Microarray profiling of gene expression is widely applied in molecular biology and functional genomics. Experimental and technical variations make meta-analysis of different studies challenging. In a total of 3358 samples, all from German population-based cohorts, we investigated the effect of data preprocessing and the variability due to sample processing in whole blood cell and blood monocyte gene expression data, measured on the Illumina HumanHT-12 v3 BeadChip array.Gene expression signal intensities were similar after applying the log(2) or the variance-stabilizing transformation. In all cohorts, the first principal component (PC) explained more than 95% of the total variation. Technical factors substantially influenced signal intensity values, especially the Illumina chip assignment (33-48% of the variance), the RNA amplification batch (12-24%), the RNA isolation batch (16%), and the sample storage time, in particular the time between blood donation and RNA isolation for the whole blood cell samples (2-3%), and the time between RNA isolation and amplification for the monocyte samples (2%). White blood cell composition parameters were the strongest biological factors influencing the expression signal intensities in the whole blood cell samples (3%), followed by sex (1-2%) in both sample types. Known single nucleotide polymorphisms (SNPs) were located in 38% of the analyzed probe sequences and 4% of them included common SNPs (minor allele frequency >5%). Out of the tested SNPs, 1.4% significantly modified the probe-specific expression signals (Bonferroni corrected p-value<0.05), but in almost half of these events the signal intensities were even increased despite the occurrence of the mismatch. Thus, the vast majority of SNPs within probes had no significant effect on hybridization efficiency.In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta

  7. Serial Analysis of 38 Proteins during the Progression of Human Breast Tumor in Mice Using an Antibody Colocalization Microarray*

    PubMed Central

    Li, Huiyan; Bergeron, Sébastien; Annis, Matthew G.; Siegel, Peter M.; Juncker, David

    2015-01-01

    Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models. PMID:25680959

  8. Progress and opportunities for tissue-engineered skin

    NASA Astrophysics Data System (ADS)

    MacNeil, Sheila

    2007-02-01

    Tissue-engineered skin is now a reality. For patients with extensive full-thickness burns, laboratory expansion of skin cells to achieve barrier function can make the difference between life and death, and it was this acute need that drove the initiation of tissue engineering in the 1980s. A much larger group of patients have ulcers resistant to conventional healing, and treatments using cultured skin cells have been devised to restart the wound-healing process. In the laboratory, the use of tissue-engineered skin provides insight into the behaviour of skin cells in healthy skin and in diseases such as vitiligo, melanoma, psoriasis and blistering disorders.

  9. Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis.

    PubMed

    Wolff, Claudia; Malinowsky, Katharina; Berg, Daniela; Schragner, Kerstin; Schuster, Tibor; Walch, Axel; Bronger, Holger; Höfler, Heinz; Becker, Karl-Friedrich

    2011-01-01

    The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.

  10. Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: Preliminary assessment of cross-platform concordance

    PubMed Central

    Kelly, Andrew D.; Hill, Katherine E.; Correll, Mick; Hu, Lan; Wang, Yaoyu; Rubio, Renee; Duan, Shenghua; Quackenbush, John; Spentzos, Dimitrios

    2014-01-01

    Next-generation sequencing is increasingly employed in biomedical investigations. Strong concordance between microarray and mRNA-seq levels has been reported in high quality specimens but information is lacking on formalin-fixed, paraffin-embedded (FFPE) tissues, and particularly for microRNA (miRNA) analysis. We conducted a preliminary examination of the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays. Quantitative agreement between platforms is moderate (Spearman correlation 0.514–0.596) and there is discordance of detection calls on a subset of miRNAs. Quantitative PCR (q-RT-PCR) performed for several discordant miRNAs confirmed the presence of most sequences detected by miRNA-seq but not by DASL but also that miRNA-seq did not detect some sequences, which DASL confidently detected. Our results suggest that miRNA-seq is specific, with few false positive calls, but it may not detect certain abundant miRNAs in FFPE tissue. Further work is necessary to fully address these issues that are pertinent for translational research. PMID:23562991

  11. Robotic multimodality stereotactic brain tissue identification: work in progress

    NASA Technical Reports Server (NTRS)

    Andrews, R.; Mah, R.; Galvagni, A.; Guerrero, M.; Papasin, R.; Wallace, M.; Winters, J.

    1997-01-01

    Real-time identification of tissue would improve procedures such as stereotactic brain biopsy (SBX), functional and implantation neurosurgery, and brain tumor excision. To standard SBX equipment has been added: (1) computer-controlled stepper motors to drive the biopsy needle/probe precisely; (2) multiple microprobes to track tissue density, detect blood vessels and changes in blood flow, and distinguish the various tissues being penetrated; (3) neural net learning programs to allow real-time comparisons of current data with a normative data bank; (4) three-dimensional graphic displays to follow the probe as it traverses brain tissue. The probe can differentiate substances such as pig brain, differing consistencies of the 'brain-like' foodstuff tofu, and gels made to simulate brain, as well as detect blood vessels imbedded in these substances. Multimodality probes should improve the safety, efficacy, and diagnostic accuracy of SBX and other neurosurgical procedures.

  12. Tendon Tissue Engineering: Progress, Challenges, and Translation to the Clinic

    PubMed Central

    Shearn, Jason T.; Kinneberg, Kirsten R.C.; Dyment, Nathaniel A.; Galloway, Marc T.; Kenter, Keith; Wylie, Christopher; Butler, David L.

    2013-01-01

    The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products. PMID:21625053

  13. Tendon tissue engineering: progress, challenges, and translation to the clinic.

    PubMed

    Shearn, J T; Kinneberg, K R; Dyment, N A; Galloway, M T; Kenter, K; Wylie, C; Butler, D L

    2011-06-01

    The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products.

  14. Tissue engineering and regenerative medicine: history, progress, and challenges.

    PubMed

    Berthiaume, François; Maguire, Timothy J; Yarmush, Martin L

    2011-01-01

    The past three decades have seen the emergence of an endeavor called tissue engineering and regenerative medicine in which scientists, engineers, and physicians apply tools from a variety of fields to construct biological substitutes that can mimic tissues for diagnostic and research purposes and can replace (or help regenerate) diseased and injured tissues. A significant portion of this effort has been translated to actual therapies, especially in the areas of skin replacement and, to a lesser extent, cartilage repair. A good amount of thoughtful work has also yielded prototypes of other tissue substitutes such as nerve conduits, blood vessels, liver, and even heart. Forward movement to clinical product, however, has been slow. Another offshoot of these efforts has been the incorporation of some new exciting technologies (e.g., microfabrication, 3D printing) that may enable future breakthroughs. In this review we highlight the modest beginnings of the field and then describe three application examples that are in various stages of development, ranging from relatively mature (skin) to ongoing proof-of-concept (cartilage) to early stage (liver). We then discuss some of the major issues that limit the development of complex tissues, some of which are fundamentals-based, whereas others stem from the needs of the end users.

  15. [Progress on cervical muscle strength and soft tissue stiffness testing].

    PubMed

    Ma, Ming; Zhang, Shi-min

    2015-08-01

    Biomechanical evaluation of neck muscles has important significance in the diagnosis and treatment for cervical spondylosis, the neck muscle strength and soft tissue stiffness test is two aspects of biomechanical testing. Isometric muscle testing operation is relatively simple, the cost is lower, which can evaluate the muscle force below grade 3. However, isokinetic muscle strength testing can assess the muscle strength of joint motion in any position. It is hard to distinguish stiffness difference in different soft tissues when the load-displacement curve is used to evaluate the local soft tissue stiffness. Elasticity imaging technique can not only show the elastic differences of different tissues by images, but also quantify the elastic modulus of subcutaneous tissues and muscles respectively. Nevertheless, it is difficult to observe the flexibility of the cervical spine by means of the analysis of the whole neck stiffness. In a word, a variety of test method will conduce not only the biomechanical evaluation of neck muscles, but also making an effective biomechanics mathematical model of neck muscles. Besides, isokinetic muscle testing and the elasticity imaging technology still need further validation and optimization before they are better applied to neck muscles biomechanical testing.

  16. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  17. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.

  18. Recent progresses in gene delivery-based bone tissue engineering.

    PubMed

    Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

    2013-12-01

    Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches. PMID:23994567

  19. Histopathological follow-up by tissue micro-array in a survival study after melanoma treated by nanosecond pulsed electric fields (nsPEF).

    PubMed

    Chen, Xinhua; Chen, Xinmei; Swanson, R James; Schoenbach, Karl H; Yin, Shengyong; Zheng, Shusen

    2011-06-01

    A recent study has shown that nanosecond pulsed electric fields (nsPEF) can affect the intracellular structures of melanoma within weeks. nsPEF is a non-drug, non-thermal treatment using ultrashort, intense pulsed electric fields with nanosecond durations. In the current study we followed up melanoma histopathology and metastasis with tissue micro-array 5 months post-nsPEF. After nsPEF treatment, tumor growth, tumor histology, metastasis, peri-tumor vessel and micro-vessel density were examined for the effect of nsPEF treatment on melanoma in vivo. The 17 nsPEF-treated mice were tumor-free for 169 days, significantly longer than those 19 control mice bearing melanoma without nsPEF. Histopathology follow-up showed that melanoma did not recur to the primary injection place after complete elimination. Compared with the control tumor, nsPEF-treated tumors present decreased micro-vessel density in a time-course manner in this survival study. Treatment with nsPEF caused continuous histopathological changes in melanomas, eliminated melanoma without recurrence at the primary site and prolonged animal survival time by inhibiting tumor blood supply and leading to tumor infarction. Thus, nsPEF could be applied in a non-ionizing therapeutic approach, without other agents, to locally treat tumors within a defined boundary.

  20. Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays.

    PubMed

    García, Juan F; Camacho, Francisca I; Morente, Manuel; Fraga, Máximo; Montalbán, Carlos; Alvaro, Tomás; Bellas, Carmen; Castaño, Angel; Díez, Ana; Flores, Teresa; Martin, Carmen; Martinez, Miguel A; Mazorra, Francisco; Menárguez, Javier; Mestre, Maria J; Mollejo, Manuela; Sáez, Ana I; Sánchez, Lydia; Piris, Miguel A

    2003-01-15

    Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.

  1. RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland.

    PubMed

    McDonald, Laura; Ferrari, Nicola; Terry, Anne; Bell, Margaret; Mohammed, Zahra M; Orange, Clare; Jenkins, Alma; Muller, William J; Gusterson, Barry A; Neil, James C; Edwards, Joanne; Morris, Joanna S; Cameron, Ewan R; Blyth, Karen

    2014-05-01

    RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.

  2. Development of an immunohistochemical protein quantification system in conjunction with tissue microarray technology for identifying predictive biomarkers for phosphatidylinositol 3-kinase inhibitors.

    PubMed

    Isoyama, Sho; Yoshimi, Hisashi; Dan, Shingo; Okamura, Mutsumi; Seki, Mariko; Irimura, Tatsuro; Yamori, Takao

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials. PMID:22975517

  3. COMPARISON OF TRANSCRIPTIONAL RESONSES FROM AVIAN GUT TISSUES AFTER EIMERIA ACERVULINA AND E. MAXIMA INFECTIONS USING cDNA MICROARRAY TECHNOLOGY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the host response during pathogen infection will extend our knowledge of pathogenesis and enhance the development of novel preventive methodologies against important infectious diseases. Microarray technology is a powerful tool to analyze host transcriptional responses. Coccidiosis re...

  4. A comprehensive immunophenotypic marker analysis of hairy cell leukemia in paraffin-embedded bone marrow trephine biopsies--a tissue microarray study.

    PubMed

    Tóth-Lipták, Judit; Piukovics, Klára; Borbényi, Zita; Demeter, Judit; Bagdi, Enikő; Krenács, László

    2015-01-01

    Hairy cell leukemia (HCL) is an uncommon B cell lymphoproliferation characterized by a unique immunophenotype. Due to low number of circulating neoplastic cells and 'dry tap' aspiration, the diagnosis is often based on BM trephine biopsy. We have performed a consecutive immunohistochemical analysis to evaluate diagnostic usefulness of various HCL markers (CD11c, CD25, CD68, CD103, CD123, CD200, annexin A1, cyclin D1, DBA.44, HBME-1, phospho-ERK1/2, TRAP, and T-bet) currently available against fixation resistant epitopes. We analyzed tissue microarrays consisting of samples gained from 73 small B-cell lymphoma cases, including hairy cell leukemia (HCL) (n = 32), HCL variant (HCL-v) (n = 4), B-cell chronic lymphocytic leukemia (B-CLL) (n = 11), lymphoplasmacytic lymphoma (LPL) (n = 3), mantle cell lymphoma (MCL) (n = 10), splenic diffuse red pulp small B cell lymphoma (SDRPL) (n = 2), splenic B cell marginal zone lymphoma (SMZL) (n = 8), and splenic B cell lymphoma/leukemia, unclassifiable (SBCL) (n = 3) cases. The HCL cases were 100% positive for all but 2 (DBA.44 and CD123) of these markers. Annexin A1 showed 100% specificity and accuracy, which was followed by CD123, pERK, CD103, HBME-1, CD11c, CD25, CD68, cyclin D1, CD200, T-bet, DBA.44, and TRAP, in decreasing order. In conclusion, our results reassured the high specificity of annexin A1 and pERK, as well as the diagnostic value of standard HCL markers of CD11c, CD25, CD103, and CD123 also in paraffin-embedded BM samples. Additional markers, including HBME-1, cyclin D1, CD200, and T-bet also represent valuable tools in the differential diagnosis of HCL and its mimics.

  5. Applications of condensed matter understanding to medical tissues and disease progression: Elemental analysis and structural integrity of tissue scaffolds

    NASA Astrophysics Data System (ADS)

    Bradley, D. A.; Farquharson, M. J.; Gundogdu, O.; Al-Ebraheem, Alia; Che Ismail, Elna; Kaabar, W.; Bunk, O.; Pfeiffer, F.; Falkenberg, G.; Bailey, M.

    2010-02-01

    The investigations reported herein link tissue structure and elemental presence with issues of environmental health and disease, exemplified by uptake and storage of potentially toxic elements in the body, the osteoarthritic condition and malignancy in the breast and other soft tissues. Focus is placed on application of state-of-the-art ionizing radiation techniques, including, micro-synchrotron X-ray fluorescence (μ-SXRF) and particle-induced X-ray emission/Rutherford backscattering mapping (μ-PIXE/RBS), coherent small-angle X-ray scattering (cSAXS) and X-ray phase-contrast imaging, providing information on elemental make-up, the large-scale organisation of collagen and anatomical features of moderate and low atomic number media. For the particular situations under investigation, use of such facilities is allowing information to be obtained at an unprecedented level of detail, yielding new understanding of the affected tissues and the progression of disease.

  6. Aptamer Microarrays

    SciTech Connect

    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

  7. Microarray analysis of thioacetamide-treated type 1 diabetic rats

    SciTech Connect

    Devi, Sachin S.; Mehendale, Harihara M. . E-mail: mehendale@ulm.edu

    2006-04-01

    It is well known that diabetes imparts high sensitivity to numerous hepatotoxicants. Previously, we have shown that a normally non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats due to inhibited tissue repair allowing progression of liver injury. On the other hand, DB rats exposed to 30 mg TA/kg exhibit delayed tissue repair and delayed recovery from injury. The objective of this study was to investigate the mechanism of impaired tissue repair and progression of liver injury in TA-treated DB rats by using cDNA microarray. Gene expression pattern was examined at 0, 6, and 12 h after TA challenge, and selected mechanistic leads from microarray experiments were confirmed by real-time RT-PCR and further investigated at protein level over the time course of 0 to 36 h after TA treatment. Diabetic condition itself increased gene expression of proteases and decreased gene expression of protease inhibitors. Administration of 300 mg TA/kg to DB rats further elevated gene expression of proteases and suppressed gene expression of protease inhibitors, explaining progression of liver injury in DB rats after TA treatment. Inhibited expression of genes involved in cell division cycle (cyclin D1, IGFBP-1, ras, E2F) was observed after exposure of DB rats to 300 mg TA/kg, explaining inhibited tissue repair in these rats. On the other hand, DB rats receiving 30 mg TA/kg exhibit delayed expression of genes involved in cell division cycle, explaining delayed tissue repair in these rats. In conclusion, impaired cyclin D1 signaling along with increased proteases and decreased protease inhibitors may explain impaired tissue repair that leads to progression of liver injury initiated by TA in DB rats.

  8. Tissue transglutaminase-2 promotes gastric cancer progression via the ERK1/2 pathway

    PubMed Central

    Zhou, Quan; Wu, Xiongyan; Chen, Xuehua; Li, Jianfang; Zhu, Zhenggang; Liu, Bingya; Su, Liping

    2016-01-01

    Gastric cancer (GC) is one of the most common tumors worldwide and involves extensive local tumor invasion, metastasis, and poor prognosis. Understanding mechanisms regulating progression of GC is necessary for developing effective therapeutic strategies. Tissue transglutaminase-2 (TG2), a multifunctional member of the transglutaminase family, has been shown to be critical for tumor initiation and progression. However, how TG2 promotes the progression of GC is unknown. We report that TG2 was highly expressed in GC tissues and positively associated with depth of tumor invasion and late TNM stage. With gain- and loss-of-function approaches, we observed that TG2 promoted GC cell proliferation, migration, invasion, as well as tumorigenesis and peritoneal metastasis in vivo. These events were associated with the ERK1/2 pathway activation and an ERK1/2 inhibitor (U0126) inhibited cell proliferation, migration, and invasion induced by overexpression of TG2. In summary, TG2 contributes to tumorigenesis and progression of GC by activating the ERK1/2 signaling pathway and is a potential therapeutic target of metastatic gastric cancer. PMID:26771235

  9. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    NASA Astrophysics Data System (ADS)

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal

    2016-02-01

    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  10. Microarrays, Integrated Analytical Systems

    NASA Astrophysics Data System (ADS)

    Combinatorial chemistry is used to find materials that form sensor microarrays. This book discusses the fundamentals, and then proceeds to the many applications of microarrays, from measuring gene expression (DNA microarrays) to protein-protein interactions, peptide chemistry, carbodhydrate chemistry, electrochemical detection, and microfluidics.

  11. Identifying Cancer Biomarkers From Microarray Data Using Feature Selection and Semisupervised Learning

    PubMed Central

    Maulik, Ujjwal

    2014-01-01

    Microarrays have now gone from obscurity to being almost ubiquitous in biological research. At the same time, the statistical methodology for microarray analysis has progressed from simple visual assessments of results to novel algorithms for analyzing changes in expression profiles. In a micro-RNA (miRNA) or gene-expression profiling experiment, the expression levels of thousands of genes/miRNAs are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on their expressions. Microarray-based gene expression profiling can be used to identify genes, whose expressions are changed in response to pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues. Recent studies have revealed that patterns of altered microarray expression profiles in cancer can serve as molecular biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and prediction of therapeutic responses. Microarray data sets containing expression profiles of a number of miRNAs or genes are used to identify biomarkers, which have dysregulation in normal and malignant tissues. However, small sample size remains a bottleneck to design successful classification methods. On the other hand, adequate number of microarray data that do not have clinical knowledge can be employed as additional source of information. In this paper, a combination of kernelized fuzzy rough set (KFRS) and semisupervised support vector machine (S3VM) is proposed for predicting cancer biomarkers from one miRNA and three gene expression data sets. Biomarkers are discovered employing three feature selection methods, including KFRS. The effectiveness of the proposed KFRS and S3VM combination on the microarray data sets is demonstrated, and the cancer biomarkers identified from miRNA data are reported. Furthermore, biological significance tests are conducted for miRNA cancer biomarkers. PMID:27170887

  12. Identifying Cancer Biomarkers From Microarray Data Using Feature Selection and Semisupervised Learning.

    PubMed

    Chakraborty, Debasis; Maulik, Ujjwal

    2014-01-01

    Microarrays have now gone from obscurity to being almost ubiquitous in biological research. At the same time, the statistical methodology for microarray analysis has progressed from simple visual assessments of results to novel algorithms for analyzing changes in expression profiles. In a micro-RNA (miRNA) or gene-expression profiling experiment, the expression levels of thousands of genes/miRNAs are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on their expressions. Microarray-based gene expression profiling can be used to identify genes, whose expressions are changed in response to pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues. Recent studies have revealed that patterns of altered microarray expression profiles in cancer can serve as molecular biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and prediction of therapeutic responses. Microarray data sets containing expression profiles of a number of miRNAs or genes are used to identify biomarkers, which have dysregulation in normal and malignant tissues. However, small sample size remains a bottleneck to design successful classification methods. On the other hand, adequate number of microarray data that do not have clinical knowledge can be employed as additional source of information. In this paper, a combination of kernelized fuzzy rough set (KFRS) and semisupervised support vector machine (S(3)VM) is proposed for predicting cancer biomarkers from one miRNA and three gene expression data sets. Biomarkers are discovered employing three feature selection methods, including KFRS. The effectiveness of the proposed KFRS and S(3)VM combination on the microarray data sets is demonstrated, and the cancer biomarkers identified from miRNA data are reported. Furthermore, biological significance tests are conducted for miRNA cancer biomarkers.

  13. 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.

  14. Current Progress in Tissue Engineering of Heart Valves: Multiscale Problems, Multiscale Solutions

    PubMed Central

    Cheung, Daniel Y; Duan, Bin; Butcher, Jonathan T.

    2016-01-01

    Introduction Heart valve disease is an increasingly prevalent and clinically serious condition. There are no clinically effective biological diagnostics or treatment strategies. The only recourse available is replacement with a prosthetic valve, but the inability of these devices to grow or respond biologically to their environments necessitates multiple resizing surgeries and life-long coagulation treatment, especially in children. Tissue engineering has a unique opportunity to impact heart valve disease by providing a living valve conduit, capable of growth and biological integration. Areas covered This review will cover current tissue engineering strategies in fabricating heart valves and their progress towards the clinic, including molded scaffolds using naturally-derived or synthetic polymers, decellularization, electrospinning, 3D bioprinting, hybrid techniques, and in vivo engineering. Expert opinion While much progress has been made to create functional living heart valves, a clinically viable product is not yet realized. The next leap in engineered living heart valves will require a deeper understanding of how the natural multi-scale structural and biological heterogeneity of the tissue ensures its efficient function. Related, improved fabrication strategies must be developed that can replicate this de novo complexity, which is likely instructive for appropriate cell differentiation and remodeling whether seeded with autologous stem cells in vitro or endogenously recruited cells. PMID:26027436

  15. Microarray analysis in pulmonary hypertension.

    PubMed

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea; Kwapiszewska, Grazyna

    2016-07-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance. PMID:27076594

  16. Microarray analysis in pulmonary hypertension

    PubMed Central

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea

    2016-01-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance. PMID:27076594

  17. IMPROVING THE RELIABILITY OF MICROARRAYS FOR TOXICOLOGY RESEARCH: A COLLABORATIVE APPROACH

    EPA Science Inventory

    Microarray-based gene expression profiling is a critical tool to identify molecular biomarkers of specific chemical stressors. Although current microarray technologies have progressed from their infancy, biological and technical repeatability and reliability are often still limit...

  18. Microarrays in hematology.

    PubMed

    Walker, Josef; Flower, Darren; Rigley, Kevin

    2002-01-01

    Microarrays are fast becoming routine tools for the high-throughput analysis of gene expression in a wide range of biologic systems, including hematology. Although a number of approaches can be taken when implementing microarray-based studies, all are capable of providing important insights into biologic function. Although some technical issues have not been resolved, microarrays will continue to make a significant impact on hematologically important research. PMID:11753074

  19. Microarrays, antiobesity and the liver

    PubMed Central

    Castro-Chávez, Fernando

    2013-01-01

    In this review, the microarray technology and especially oligonucleotide arrays are exemplified with a practical example taken from the perilipin−/− mice and using the dChip software, available for non-lucrative purposes. It was found that the liver of perilipin−/− mice was healthy and normal, even under high-fat diet when compared with the results published for the scd1−/− mice, which under high-fat diets had a darker liver, suggestive of hepatic steatosis. Scd1 is required for the biosynthesis of monounsaturated fatty acids and plays a key role in the hepatic synthesis of triglycerides and of very-low-density lipoproteins. Both models of obesity resistance share many similar phenotypic antiobesity features, however, the perilipin−/− mice had a significant downregulation of stearoyl CoA desaturases scd1 and scd2 in its white adipose tissue, but a normal level of both genes inside the liver, even under high-fat diet. Here, different microarray methodologies are discussed, and also some of the most recent discoveries and perspectives regarding the use of microarrays, with an emphasis on obesity gene expression, and a personal remark on my findings of increased expression for hemoglobin transcripts and other hemo related genes (hemo-like), and for leukocyte like (leuko-like) genes inside the white adipose tissue of the perilipin−/− mice. In conclusion, microarrays have much to offer in comparative studies such as those in antiobesity, and also they are methodologies adequate for new astounding molecular discoveries [free full text of this article PMID:15657555

  20. Pineal Function: Impact of Microarray Analysis

    PubMed Central

    Klein, David C.; Bailey, Michael J.; Carter, David A.; Kim, Jong-so; Shi, Qiong; Ho, Anthony; Chik, Constance; Gaildrat, Pascaline; Morin, Fabrice; Ganguly, Surajit; Rath, Martin F.; Møller, Morten; Sugden, David; Rangel, Zoila G.; Munson, Peter J.; Weller, Joan L.; Coon, Steven L.

    2009-01-01

    Microarray analysis has provided a new understanding of pineal function by identifying genes that are highly expressed in this tissue relative to other tissues and also by identifying over 600 genes that are expressed on a 24-hour schedule. This effort has highlighted surprising similarity to the retina and has provided reason to explore new avenues of study including intracellular signaling, signal transduction, transcriptional cascades, thyroid/retinoic acid hormone signaling, metal biology, RNA splicing, and the role the pineal gland plays in the immune/inflammation response. The new foundation that microarray analysis has provided will broadly support future research on pineal function. PMID:19622385

  1. MicroRNA expression profiling using microarrays.

    PubMed

    Love, Cassandra; Dave, Sandeep

    2013-01-01

    MicroRNAs are small noncoding RNAs which are able to regulate gene expression at both the transcriptional and translational levels. There is a growing recognition of the role of microRNAs in nearly every tissue type and cellular process. Thus there is an increasing need for accurate quantitation of microRNA expression in a variety of tissues. Microarrays provide a robust method for the examination of microRNA expression. In this chapter, we describe detailed methods for the use of microarrays to measure microRNA expression and discuss methods for the analysis of microRNA expression data. PMID:23666707

  2. Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

    PubMed Central

    Pondeville, Emilie; David, Jean-Philippe; Guittard, Emilie; Maria, Annick; Ranson, Hilary

    2013-01-01

    In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the “black box”, remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae. PMID:24324583

  3. [Connective tissue growth factor (CTGF): a key factor in the onset and progression of kidney damage].

    PubMed

    Sánchez-López, E; Rodrigues Díez, R; Rodríguez Vita, J; Rayego Mateos, S; Rodrigues Díez, R R; Rodríguez García, E; Lavoz Barria, C; Mezzano, S; Egido, J; Ortiz, A; Ruiz-Ortega, M; Selgas, R

    2009-01-01

    Connective tissue growth factor (CTGF) is increased in several pathologies associated with fibrosis, including multiple renal diseases. CTGF is involved in biological processes such as cell cycle regulation, migration, adhesion and angiogenesis. Its expression is regulated by various factors involved in renal damage, such as transforming growth factor- , Angiotensin II, high concentrations of glucose and cellular stress. CTGF is involved in the initiation and progression of renal damage to be able to induce an inflammatory response and promote fibrosis, identified as a potential therapeutic target in the treatment of kidney diseases. In this paper we review the main actions of CTGF in renal disease, the intracellular action mechanisms and therapeutic strategies for its blocking.

  4. Antiphospholipid antibodies promote tissue factor-dependent angiogenic switch and tumor progression.

    PubMed

    Wu, Yuan-Yuan; V Nguyen, Andrew; Wu, Xiao-Xuan; Loh, Mingyu; Vu, Michelle; Zou, Yiyu; Liu, Qiang; Guo, Peng; Wang, Yanhua; Montgomery, Leslie L; Orlofsky, Amos; Rand, Jacob H; Lin, Elaine Y

    2014-12-01

    Progression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell-derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism.

  5. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression

    PubMed Central

    Laklai, Hanane; Miroshnikova, Yekaterina A.; Pickup, Michael W.; Collisson, Eric A.; Kim, Grace E.; Barrett, Alex S.; Hill, Ryan C.; Lakins, Johnathon N.; Schlaepfer, David D.; Mouw, Janna K.; LeBleu, Valerie S.; Roy, Nilotpal; Novitskiy, Sergey V.; Johansen, Julia S.; Poli, Valeria; Kalluri, Raghu; Iacobuzio-Donahue, Christine A.; Wood, Laura D.; Hebrok, Matthias; Hansen, Kirk; Moses, Harold L.; Weaver, Valerie M.

    2016-01-01

    Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype. PMID:27089513

  6. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.

    PubMed

    Laklai, Hanane; Miroshnikova, Yekaterina A; Pickup, Michael W; Collisson, Eric A; Kim, Grace E; Barrett, Alex S; Hill, Ryan C; Lakins, Johnathon N; Schlaepfer, David D; Mouw, Janna K; LeBleu, Valerie S; Roy, Nilotpal; Novitskiy, Sergey V; Johansen, Julia S; Poli, Valeria; Kalluri, Raghu; Iacobuzio-Donahue, Christine A; Wood, Laura D; Hebrok, Matthias; Hansen, Kirk; Moses, Harold L; Weaver, Valerie M

    2016-05-01

    Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

  7. Genomic-Wide Analysis with Microarrays in Human Oncology

    PubMed Central

    Inaoka, Kenichi; Inokawa, Yoshikuni; Nomoto, Shuji

    2015-01-01

    DNA microarray technologies have advanced rapidly and had a profound impact on examining gene expression on a genomic scale in research. This review discusses the history and development of microarray and DNA chip devices, and specific microarrays are described along with their methods and applications. In particular, microarrays have detected many novel cancer-related genes by comparing cancer tissues and non-cancerous tissues in oncological research. Recently, new methods have been in development, such as the double-combination array and triple-combination array, which allow more effective analysis of gene expression and epigenetic changes. Analysis of gene expression alterations in precancerous regions compared with normal regions and array analysis in drug-resistance cancer tissues are also successfully performed. Compared with next-generation sequencing, a similar method of genome analysis, several important differences distinguish these techniques and their applications. Development of novel microarray technologies is expected to contribute to further cancer research.

  8. Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression.

    PubMed

    Heissig, Beate; Dhahri, Douaa; Eiamboonsert, Salita; Salama, Yousef; Shimazu, Hiroshi; Munakata, Shinya; Hattori, Koichi

    2015-12-01

    Tissue regeneration during wound healing or cancer growth and progression depends on the establishment of a cellular microenvironment. Mesenchymal stem cells (MSC) are part of this cellular microenvironment, where they functionally modulate cell homing, angiogenesis, and immune modulation. MSC recruitment involves detachment of these cells from their niche, and finally MSC migration into their preferred niches; the wounded area, the tumor bed, and the BM, just to name a few. During this recruitment phase, focal proteolysis disrupts the extracellular matrix (ECM) architecture, breaks cell-matrix interactions with receptors, and integrins, and causes the release of bioactive fragments from ECM molecules. MSC produce a broad array of proteases, promoting remodeling of the surrounding ECM through proteolytic mechanisms. The fibrinolytic system, with its main player plasmin, plays a crucial role in cell migration, growth factor bioavailability, and the regulation of other protease systems during inflammation, tissue regeneration, and cancer. Key components of the fibrinolytic cascade, including the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are expressed in MSC. This review will introduce general functional properties of the fibrinolytic system, which go beyond its known function of fibrin clot dissolution (fibrinolysis). We will focus on the role of the fibrinolytic system for MSC biology, summarizing our current understanding of the role of the fibrinolytic system for MSC recruitment and the functional consequences for tissue regeneration and cancer. Aspects of MSC origin, maintenance, and the mechanisms by which these cells contribute to altered protease activity in the microenvironment under normal and pathological conditions will also be discussed.

  9. Microarrays in Glycoproteomics Research

    PubMed Central

    Yue, Tingting; Haab, Brian B.

    2009-01-01

    Microarrays have been extremely useful for investigating binding interactions among diverse types of molecular species, with the main advantage being the ability to examine many interactions using small amount of samples and reagents. Microarrays are increasingly being used to advance research in the field of glycobiology, which is the study of the nature and function and carbohydrates in health and disease. Several types of microarrays are being used in the study of glycans and proteins in glycobiology, including glycan arrays to study the recognition of carbohydrates, lectin arrays to determine carbohydrate expression on purified proteins or on cells, and antibody arrays to examine the variation in particular glycan structures on specific proteins. This review will cover the technology and applications of these types of microarrays, as well as their use for obtaining complementary information on various aspects of glycobiology. PMID:19389548

  10. Microarray analysis of microRNA expression in mouse fetus at 13.5 and 14.5 days post-coitum in ear and back skin tissues.

    PubMed

    Torres, Leda; Juárez, Ulises; García, Laura; Miranda-Ríos, Juan; Frias, Sara

    2016-09-01

    There is no information regarding the role of microRNAs in the development of the external ear in mammals. The purpose of this study was to determine the stage-specific expression of microRNA during external ear development in mice under normal conditions. GeneChip miRNA 3.0 arrays by Affymetrix were used to obtain miRNA expression profiles from mice fetal pinnae and back skin tissues at 13.5 days-post-coitum (dpc) and 14.5 dpc. Biological triplicates for each tissue were analyzed; one litter represents one biological replica, each litter had 16 fetuses on average. The results were analyzed with Affymetrix's Transcriptome Analysis Console software to identify differentially expressed miRNAs. The inquiry showed significant differential expression of 25 miRNAs at 13.5 dpc and 31 at 14.5 dpc, some of these miRNAs were predicted to target genes implicated in external ear development. One example is mmu-miR-10a whose low expression in pinnae is known to impact ear development by modulating Hoxa1 mRNA levels Garzon et al. (2006), Gavalas et al. (1998) [1], [2]. Other findings like the upregulation of mmu-miR-200c and mmu-miR-205 in the pinnae tissues of healthy mice are in agreement with what has been reported in human patients with microtia, in which down regulation of both miRNAs has been found Li et al. (2013) [3]. This study uncovered a spatiotemporal pattern of miRNA expression in the external ear, which results from continuous transcriptional changes during normal development of body structures. All microarray data are available at the Gene Expression Omnibus (GEO) at NCBI under accession number GSE64945. PMID:27408816

  11. Microarray analysis of microRNA expression in mouse fetus at 13.5 and 14.5 days post-coitum in ear and back skin tissues.

    PubMed

    Torres, Leda; Juárez, Ulises; García, Laura; Miranda-Ríos, Juan; Frias, Sara

    2016-09-01

    There is no information regarding the role of microRNAs in the development of the external ear in mammals. The purpose of this study was to determine the stage-specific expression of microRNA during external ear development in mice under normal conditions. GeneChip miRNA 3.0 arrays by Affymetrix were used to obtain miRNA expression profiles from mice fetal pinnae and back skin tissues at 13.5 days-post-coitum (dpc) and 14.5 dpc. Biological triplicates for each tissue were analyzed; one litter represents one biological replica, each litter had 16 fetuses on average. The results were analyzed with Affymetrix's Transcriptome Analysis Console software to identify differentially expressed miRNAs. The inquiry showed significant differential expression of 25 miRNAs at 13.5 dpc and 31 at 14.5 dpc, some of these miRNAs were predicted to target genes implicated in external ear development. One example is mmu-miR-10a whose low expression in pinnae is known to impact ear development by modulating Hoxa1 mRNA levels Garzon et al. (2006), Gavalas et al. (1998) [1], [2]. Other findings like the upregulation of mmu-miR-200c and mmu-miR-205 in the pinnae tissues of healthy mice are in agreement with what has been reported in human patients with microtia, in which down regulation of both miRNAs has been found Li et al. (2013) [3]. This study uncovered a spatiotemporal pattern of miRNA expression in the external ear, which results from continuous transcriptional changes during normal development of body structures. All microarray data are available at the Gene Expression Omnibus (GEO) at NCBI under accession number GSE64945.

  12. DNA Microarray Technology

    SciTech Connect

    WERNER-WASHBURNE, MARGARET; DAVIDSON, GEORGE S.

    2002-01-01

    Collaboration between Sandia National Laboratories and the University of New Mexico Biology Department resulted in the capability to train students in microarray techniques and the interpretation of data from microarray experiments. These studies provide for a better understanding of the role of stationary phase and the gene regulation involved in exit from stationary phase, which may eventually have important clinical implications. Importantly, this research trained numerous students and is the basis for three new Ph.D. projects.

  13. Photo-Generation of Carbohydrate Microarrays

    NASA Astrophysics Data System (ADS)

    Carroll, Gregory T.; Wang, Denong; Turro, Nicholas J.; Koberstein, Jeffrey T.

    The unparalleled structural diversity of carbohydrates among biological molecules has been recognized for decades. Recent studies have highlighted carbohydrate signaling roles in many important biological processes, such as fertilization, embryonic development, cell differentiation and cellȁ4cell communication, blood coagulation, inflammation, chemotaxis, as well as host recognition and immune responses to microbial pathogens. In this chapter, we summarize recent progress in the establishment of carbohydrate-based microarrays and the application of these technologies in exploring the biological information content in carbohydrates. A newly established photochemical platform of carbohydrate microarrays serves as a model for a focused discussion.

  14. Protein Microarrays for the Detection of Biothreats

    NASA Astrophysics Data System (ADS)

    Herr, Amy E.

    Although protein microarrays have proven to be an important tool in proteomics research, the technology is emerging as useful for public health and defense applications. Recent progress in the measurement and characterization of biothreat agents is reviewed in this chapter. Details concerning validation of various protein microarray formats, from contact-printed sandwich assays to supported lipid bilayers, are presented. The reviewed technologies have important implications for in vitro characterization of toxin-ligand interactions, serotyping of bacteria, screening of potential biothreat inhibitors, and as core components of biosensors, among others, research and engineering applications.

  15. DNA microarrays in neuropsychopharmacology.

    PubMed

    Marcotte, E R; Srivastava, L K; Quirion, R

    2001-08-01

    Recent advances in experimental genomics, coupled with the wealth of sequence information available for a variety of organisms, have the potential to transform the way pharmacological research is performed. At present, high-density DNA microarrays allow researchers to quickly and accurately quantify gene-expression changes in a massively parallel manner. Although now well established in other biomedical fields, such as cancer and genetics research, DNA microarrays have only recently begun to make significant inroads into pharmacology. To date, the major focus in this field has been on the general application of DNA microarrays to toxicology and drug discovery and design. This review summarizes the major microarray findings of relevance to neuropsychopharmacology, as a prelude to the design and analysis of future basic and clinical microarray experiments. The ability of DNA microarrays to monitor gene expression simultaneously in a large-scale format is helping to usher in a post-genomic age, where simple constructs about the role of nature versus nurture are being replaced by a functional understanding of gene expression in living organisms. PMID:11479006

  16. [Research progress in peri-implant soft tissue engineering augmentation method].

    PubMed

    Pei, Tingting; Yu, Hongqiang; Wen, Chaoju; Guo, Tianqi; Zhou, Yanmin; Peng, Huimin

    2016-05-01

    The sufficiency of hard and soft tissue at the implant site is the guarantee of long-term function, health and the appearance of implant denture. Problem of soft tissue recession at the implant site has always been bothering dentists. Traditional methods for augmentation of soft tissue such as gingival transplantation have disadvantages of instability of the increased soft-tissue and more trauma. Lately the methods that base on tissue engineering to increase the soft tissue of peri-implant sites have drawn great attention. This review focuses on the current methods of peri-implant restoration through tissue engineering, seed cells, biological scaffolds and cytokines. PMID:27220393

  17. 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

  18. High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses.

    PubMed

    Abd El-Rehim, Dalia M; Ball, Graham; Pinder, Sarah E; Rakha, Emad; Paish, Claire; Robertson, John F R; Macmillan, Douglas; Blamey, Roger W; Ellis, Ian O

    2005-09-01

    Recent studies on gene molecular profiling using cDNA microarray in a relatively small series of breast cancer have identified biologically distinct groups with apparent clinical and prognostic relevance. The validation of such new taxonomies should be confirmed on larger series of cases prior to acceptance in clinical practice. The development of tissue microarray (TMA) technology provides methodology for high-throughput concomitant analyses of multiple proteins on large numbers of archival tumour samples. In our study, we have used immunohistochemistry techniques applied to TMA preparations of 1,076 cases of invasive breast cancer to study the combined protein expression profiles of a large panel of well-characterized commercially available biomarkers related to epithelial cell lineage, differentiation, hormone and growth factor receptors and gene products known to be altered in some forms of breast cancer. Using hierarchical clustering methodology, 5 groups with distinct patterns of protein expression were identified. A sixth group of only 4 cases was also identified but deemed too small for further detailed assessment. Further analysis of these clusters was performed using multiple layer perceptron (MLP)-artificial neural network (ANN) with a back propagation algorithm to identify key biomarkers driving the membership of each group. We have identified 2 large groups by their expression of luminal epithelial cell phenotypic characteristics, hormone receptors positivity, absence of basal epithelial phenotype characteristics and lack of c-erbB-2 protein overexpression. Two additional groups were characterized by high c-erbB-2 positivity and negative or weak hormone receptors expression but showed differences in MUC1 and E-cadherin expression. The final group was characterized by strong basal epithelial characteristics, p53 positivity, absent hormone receptors and weak to low luminal epithelial cytokeratin expression. In addition, we have identified significant

  19. Hidden Treasures in “Ancient” Microarrays: Gene-Expression Portrays Biology and Potential Resistance Pathways of Major Lung Cancer Subtypes and Normal Tissue

    PubMed Central

    Kerkentzes, Konstantinos; Lagani, Vincenzo; Tsamardinos, Ioannis; Vyberg, Mogens; Røe, Oluf Dimitri

    2014-01-01

    Objective: Novel statistical methods and increasingly more accurate gene annotations can transform “old” biological data into a renewed source of knowledge with potential clinical relevance. Here, we provide an in silico proof-of-concept by extracting novel information from a high-quality mRNA expression dataset, originally published in 2001, using state-of-the-art bioinformatics approaches. Methods: The dataset consists of histologically defined cases of lung adenocarcinoma (AD), squamous (SQ) cell carcinoma, small-cell lung cancer, carcinoid, metastasis (breast and colon AD), and normal lung specimens (203 samples in total). A battery of statistical tests was used for identifying differential gene expressions, diagnostic and prognostic genes, enriched gene ontologies, and signaling pathways. Results: Our results showed that gene expressions faithfully recapitulate immunohistochemical subtype markers, as chromogranin A in carcinoids, cytokeratin 5, p63 in SQ, and TTF1 in non-squamous types. Moreover, biological information with putative clinical relevance was revealed as potentially novel diagnostic genes for each subtype with specificity 93–100% (AUC = 0.93–1.00). Cancer subtypes were characterized by (a) differential expression of treatment target genes as TYMS, HER2, and HER3 and (b) overrepresentation of treatment-related pathways like cell cycle, DNA repair, and ERBB pathways. The vascular smooth muscle contraction, leukocyte trans-endothelial migration, and actin cytoskeleton pathways were overexpressed in normal tissue. Conclusion: Reanalysis of this public dataset displayed the known biological features of lung cancer subtypes and revealed novel pathways of potentially clinical importance. The findings also support our hypothesis that even old omics data of high quality can be a source of significant biological information when appropriate bioinformatics methods are used. PMID:25325012

  20. Comparison of hepatocellular carcinoma miRNA expression profiling as evaluated by next generation sequencing and microarray.

    PubMed

    Murakami, Yoshiki; Tanahashi, Toshihito; Okada, Rina; Toyoda, Hidenori; Kumada, Takashi; Enomoto, Masaru; Tamori, Akihiro; Kawada, Norifumi; Taguchi, Y-h; Azuma, Takeshi

    2014-01-01

    MicroRNA (miRNA) expression profiling has proven useful in diagnosing and understanding the development and progression of several diseases. Microarray is the standard method for analyzing miRNA expression profiles; however, it has several disadvantages, including its limited detection of miRNAs. In recent years, advances in genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which significantly advance genome sequencing speed and discovery. In this study, we compared the expression profiles obtained by next generation sequencing (NGS) with the profiles created using microarray to assess if NGS could produce a more accurate and complete miRNA profile. Total RNA from 14 hepatocellular carcinoma tumors (HCC) and 6 matched non-tumor control tissues were sequenced with Illumina MiSeq 50-bp single-end reads. Micro RNA expression profiles were estimated using miRDeep2 software. As a comparison, miRNA expression profiles for 11 out of 14 HCCs were also established by microarray (Agilent human microRNA microarray). The average total sequencing exceeded 2.2 million reads per sample and of those reads, approximately 57% mapped to the human genome. The average correlation for miRNA expression between microarray and NGS and subtraction were 0.613 and 0.587, respectively, while miRNA expression between technical replicates was 0.976. The diagnostic accuracy of HCC, p-value, and AUC were 90.0%, 7.22×10(-4), and 0.92, respectively. In summary, NGS created an miRNA expression profile that was reproducible and comparable to that produced by microarray. Moreover, NGS discovered novel miRNAs that were otherwise undetectable by microarray. We believe that miRNA expression profiling by NGS can be a useful diagnostic tool applicable to multiple fields of medicine.

  1. Applications of Functional Protein Microarrays in Basic and Clinical Research

    PubMed Central

    Zhu, Heng; Qian, Jiang

    2013-01-01

    The protein microarray technology provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. It is viewed as a new tool that overcomes the limitation of DNA microarrays. On the basis of its application, protein microarrays fall into two major classes: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be directly spotted on a slide to form the so-called “reverse-phase” protein microarray. In the last decade, applications of functional protein microarrays in particular have flourished in studying protein function and construction of networks and pathways. In this chapter, we will review the recent advancements in the protein microarray technology, followed by presenting a series of examples to illustrate the power and versatility of protein microarrays in both basic and clinical research. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:22989767

  2. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  3. MicroRNA-203 inhibits the progression of esophageal squamous cell carcinoma with restored epithelial tissue architecture in vivo.

    PubMed

    Okumura, Tomoyuki; Shimada, Yutaka; Moriyama, Makoto; Takei, Yoshinori; Omura, Tetsuya; Sekine, Shinichi; Nagata, Takuya; Shimizu, Kazuharu; Tsukada, Kazuhiro

    2014-06-01

    MicroRNA (miR)-203 has been shown to induce squamous differentiation of epidermal stem cells through the suppression of p63. The aim of this study was to assess the tumor suppressor effect of miR-203 in esophageal squamous cell carcinoma (ESCC) with focus on the regulation of the cell fate decisions and organization of tumor tissue architecture in vivo. Our investigation establishing stable clones from ESCC cell lines with induced miR-203 expression resulted in significant growth inhibition in a mouse xenograft model. Small foci were observed in xenograft tumors with stratified squamous differentiation in conjunction with restored baso-apical polarity. The expression of the basement membrane protein laminine was localized at the center of the foci and the basal cell marker p75NTR was expressed in the innermost layer. The expression of ki67 and p63 was co-localized at the center layers, while involucrin was expressed in the outer layers. Flow cytometry revealed that the p75NTR-positive cells expressing p63 and Bmi1 were well maintained, while the expression of p63 was suppressed in the p75NTR-negative cells. Our cDNA microarray analysis demonstrated the upregulation of genes involved in regulating tissue architecture, such as BMP-4 and ZO-1 in the mir-203 transfectant. Investigation using surgically removed ESCC specimens revealed that the expression of miR-203 significantly correlated with a favorable prognosis. These results demonstrated that miR-203 regulated both basal and supra-basal cell components to induce differentiation with restored epithelial tissue architecture, leading to significant tumor growth inhibition in vivo. Those results suggest the use of miR-203 as a novel therapeutic and diagnostic target in patients with ESCC.

  4. Early Prediction of Cancer Progression by Depth-Resolved Nanoscale Mapping of Nuclear Architecture from Unstained Tissue Specimens.

    PubMed

    Uttam, Shikhar; Pham, Hoa V; LaFace, Justin; Leibowitz, Brian; Yu, Jian; Brand, Randall E; Hartman, Douglas J; Liu, Yang

    2015-11-15

    Early cancer detection currently relies on screening the entire at-risk population, as with colonoscopy and mammography. Therefore, frequent, invasive surveillance of patients at risk for developing cancer carries financial, physical, and emotional burdens because clinicians lack tools to accurately predict which patients will actually progress into malignancy. Here, we present a new method to predict cancer progression risk via nanoscale nuclear architecture mapping (nanoNAM) of unstained tissue sections based on the intrinsic density alteration of nuclear structure rather than the amount of stain uptake. We demonstrate that nanoNAM detects a gradual increase in the density alteration of nuclear architecture during malignant transformation in animal models of colon carcinogenesis and in human patients with ulcerative colitis, even in tissue that appears histologically normal according to pathologists. We evaluated the ability of nanoNAM to predict "future" cancer progression in patients with ulcerative colitis who did and did not develop colon cancer up to 13 years after their initial colonoscopy. NanoNAM of the initial biopsies correctly classified 12 of 15 patients who eventually developed colon cancer and 15 of 18 who did not, with an overall accuracy of 85%. Taken together, our findings demonstrate great potential for nanoNAM in predicting cancer progression risk and suggest that further validation in a multicenter study with larger cohorts may eventually advance this method to become a routine clinical test. PMID:26383164

  5. Early Prediction of Cancer Progression by Depth-Resolved Nanoscale Mapping of Nuclear Architecture from Unstained Tissue Specimens.

    PubMed

    Uttam, Shikhar; Pham, Hoa V; LaFace, Justin; Leibowitz, Brian; Yu, Jian; Brand, Randall E; Hartman, Douglas J; Liu, Yang

    2015-11-15

    Early cancer detection currently relies on screening the entire at-risk population, as with colonoscopy and mammography. Therefore, frequent, invasive surveillance of patients at risk for developing cancer carries financial, physical, and emotional burdens because clinicians lack tools to accurately predict which patients will actually progress into malignancy. Here, we present a new method to predict cancer progression risk via nanoscale nuclear architecture mapping (nanoNAM) of unstained tissue sections based on the intrinsic density alteration of nuclear structure rather than the amount of stain uptake. We demonstrate that nanoNAM detects a gradual increase in the density alteration of nuclear architecture during malignant transformation in animal models of colon carcinogenesis and in human patients with ulcerative colitis, even in tissue that appears histologically normal according to pathologists. We evaluated the ability of nanoNAM to predict "future" cancer progression in patients with ulcerative colitis who did and did not develop colon cancer up to 13 years after their initial colonoscopy. NanoNAM of the initial biopsies correctly classified 12 of 15 patients who eventually developed colon cancer and 15 of 18 who did not, with an overall accuracy of 85%. Taken together, our findings demonstrate great potential for nanoNAM in predicting cancer progression risk and suggest that further validation in a multicenter study with larger cohorts may eventually advance this method to become a routine clinical test.

  6. Hybridization and Selective Release of DNA Microarrays

    SciTech Connect

    Beer, N R; Baker, B; Piggott, T; Maberry, S; Hara, C M; DeOtte, J; Benett, W; Mukerjee, E; Dzenitis, J; Wheeler, E K

    2011-11-29

    DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to

  7. Tissue factor associates with survival and regulates tumour progression in osteosarcoma.

    PubMed

    Tieken, Chris; Verboom, Michiel C; Ruf, Wolfram; Gelderblom, Hans; Bovée, Judith V M G; Reitsma, Pieter H; Cleton-Jansen, Anne-Marie; Versteeg, Henri H

    2016-05-01

    Osteosarcoma is the most common primary malignant bone tumour. Patients often develop lung metastasis and have a poor prognosis despite extensive chemotherapy and surgical resections. Tissue Factor is associated with poor clinical outcome in a wide range of cancer types, and promotes angiogenesis and metastasis. The role of Tissue Factor in OS tumourigenesis is unknown. Fifty-three osteosarcoma pre-treatment biopsies and four osteosarcoma cell lines were evaluated for Tissue Factor expression, and a possible association with clinical parameters was investigated. Tissue Factor function was inhibited in an osteosarcoma cell line (143B) by shRNA knockdown or specific antibodies, and pro-tumourigenic gene expression, proliferation, matrigel invasion and transwell migration was examined. 143B cells were implanted in mice in the presence of Tissue Factor-blocking antibodies, and tumour volume, micro-vessel density and metastases in the lung were evaluated. Tissue Factor was highly expressed in 73.6 % of osteosarcoma biopsies, and expression associated significantly with disease-free survival. Tissue Factor was expressed in all four investigated cell lines. Tissue Factor was knocked down in 143B cells, which led to reduced expression of IL-8, CXCL-1, SNAIL and MMP2, but not MMP9. Tissue Factor knockdown or inhibition with antibodies reduced matrigel invasion. Tissue Factor antibodies limited 143B tumour growth in vivo, and resulted in decreased intra-tumoural micro-vessel density. Furthermore, lung metastasis from the primary tumour was significantly reduced. Thus, Tissue Factor expression in osteosarcoma reduces metastasis-free survival in patients, and increases pro-tumourigenic behaviour both in vitro and in vivo. PMID:26763081

  8. Bone tissue engineering using silica-based mesoporous nanobiomaterials:Recent progress.

    PubMed

    Shadjou, Nasrin; Hasanzadeh, Mohammad

    2015-10-01

    Bone disorders are of significant concern due to increase in the median age of our population. It is in this context that tissue engineering has been emerging as a valid approach to the current therapies for bone regeneration/substitution. Tissue-engineered bone constructs have the potential to alleviate the demand arising from the shortage of suitable autograft and allograft materials for augmenting bone healing. Silica based mesostructured nanomaterials possessing pore sizes in the range 2-50 nm and surface reactive functionalities have elicited immense interest due to their exciting prospects in bone tissue engineering. In this review we describe application of silica-based mesoporous nanomaterials for bone tissue engineering. We summarize the preparation methods, the effect of mesopore templates and composition on the mesopore-structure characteristics, and different forms of these materials, including particles, fibers, spheres, scaffolds and composites. Also, the effect of structural and textural properties of mesoporous materials on development of new biomaterials for production of bone implants and bone cements was discussed. Also, application of different mesoporous materials on construction of manufacture 3-dimensional scaffolds for bone tissue engineering was discussed. It begins by giving the reader a brief background on tissue engineering, followed by a comprehensive description of all the relevant components of silica-based mesoporous biomaterials on bone tissue engineering, going from materials to scaffolds and from cells to tissue engineering strategies that will lead to "engineered" bone.

  9. [Progress and challenges in tissue engineering of intervertebral disc annulus fibrosus].

    PubMed

    Zhou, Pinghui; Guo, Qianping; Ling, Feng; Qian, Zhonglai; Li, Bin

    2016-03-01

    Degenerative disc disease (DDD) is a leading cause of low back pain, which severely affects the quality of life and incurs significant medical cost. Annulus fibrosus(AF) injuries can lead to substantial deterioration of intervertebral disc degeneration. However, the AF repair/regeneration remains a challenge due to the intrinsic cellular, biochemical and biomechanical heterogeneity of AF tissue. Tissue engineering would be a promising approach for AF regeneration. This article aims to provide a brief overview of the fundamental aspects of AF, the current achievements and future challenges of AF tissue engineering. A multidisciplinary approach is proposed for future studies to fully mimic the native AF tissue and its microenvironment, including choosing adequate cell source, preparing scaffolds with hierarchical microstructures, supplementing appropriate growth factors, and enforcing suitable mechanical stimulation. Hopefully, the engineered AF tissues would be effectively used to facilitate the treatment of DDD in the future. PMID:27273986

  10. Microarrays under the microscope

    PubMed Central

    Wildsmith, S E; Elcock, F J

    2001-01-01

    Microarray technology is a rapidly advancing area, which is gaining popularity in many biological disciplines from drug target identification to predictive toxicology. Over the past few years, there has been a dramatic increase in the number of methods and techniques available for carrying out this form of gene expression analysis. The techniques and associated peripherals, such as slide types, deposition methods, robotics, and scanning equipment, are undergoing constant improvement, helping to drive the technology forward in terms of robustness and ease of use. These rapid developments, combined with the number of options available and the associated hyperbole, can prove daunting for the new user. This review aims to guide the researcher through the various steps of conducting microarray experiments, from initial strategy to analysing the data, with critical examination of the benefits and disadvantages along the way. PMID:11212888

  11. Navigating public microarray databases.

    PubMed

    Penkett, Christopher J; Bähler, Jürg

    2004-01-01

    With the ever-escalating amount of data being produced by genome-wide microarray studies, it is of increasing importance that these data are captured in public databases so that researchers can use this information to complement and enhance their own studies. Many groups have set up databases of expression data, ranging from large repositories, which are designed to comprehensively capture all published data, through to more specialized databases. The public repositories, such as ArrayExpress at the European Bioinformatics Institute contain complete datasets in raw format in addition to processed data, whilst the specialist databases tend to provide downstream analysis of normalized data from more focused studies and data sources. Here we provide a guide to the use of these public microarray resources.

  12. Real-time optical monitoring of permanent lesion progression in radiofrequency ablated cardiac tissue (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Singh-Moon, Rajinder P.; Hendon, Christine P.

    2016-02-01

    Despite considerable advances in guidance of radiofrequency ablation (RFA) therapies for atrial fibrillation, success rates have been hampered by an inability to intraoperatively characterize the extent of permanent injury. Insufficient lesions can elusively create transient conduction blockages that eventually reconduct. Prior studies suggest significantly greater met-myoglobin (Mmb) concentrations in the lesion core than those in the healthy myocardium and may serve as a marker for irreversible tissue damage. In this work, we present real-time monitoring of permanent injury through spectroscopic assessment of Mmb concentrations at the catheter tip. Atrial wedges (n=6) were excised from four fresh swine hearts and submerged under pulsatile flow of warm (37oC) phosphate buffered saline. A commercial RFA catheter inserted into a fiber optic sheath allowed for simultaneous measurement of tissue diffuse reflectance (DR) spectra (500-650nm) during application of RF energy. Optical measurements were continuously acquired before, during, and post-ablation, in addition to healthy neighboring tissue. Met-myoglobin, oxy-myoglobin, and deoxy-myoglobin concentrations were extracted from each spectrum using an inverse Monte Carlo method. Tissue injury was validated with Masson's trichrome and hematoxylin and eosin staining. Time courses revealed a rapid increase in tissue Mmb concentrations at the onset of RFA treatment and a gradual plateauing thereafter. Extracted Mmb concentrations were significantly greater post-ablation (p<0.0001) as compared to healthy tissue and correlated well with histological assessment of severe thermal tissue destruction. On going studies are aimed at integrating these findings with prior work on near infrared spectroscopic lesion depth assessment. These results support the use of spectroscopy-facilitated guidance of RFA therapies for real-time permanent injury estimation.

  13. [Research progress on application of carbon nanotubes in bone tissue engineering scaffold].

    PubMed

    Yao, Mengzhu; Sheng, Xiaoxia; Lin, Jun; Gao, Jianqing

    2016-03-01

    Carbon nanotubes possess excellent mechanical and electrical properties and demonstrate broad application prospects in medical fields. Carbon nanotubes are composed of inorganic materials, natural biodegradable polymer or synthetic biodegradable polymer. The composite bone tissue engineering scaffolds are constructed by particle-hole method, lyophilization, microsphere aggregation method, electrostatic spinning or three-dimensional printing. Composite scaffolds overcome the shortcomings of single material and have good biocompatibility, osteoconduction and osteoinduction. With the study of surface chemistry, toxicology, and biocompatibility, a degradable "human-friendly" carbon nanotubes composite bone tissue scaffold will be available; and under the drive of new fabrication techniques, the clinical application of carbon nanotubes composite bone tissue engineering scaffolds will be better developed.

  14. [Research progress on application of carbon nanotubes in bone tissue engineering scaffold].

    PubMed

    Yao, Mengzhu; Sheng, Xiaoxia; Lin, Jun; Gao, Jianqing

    2016-03-01

    Carbon nanotubes possess excellent mechanical and electrical properties and demonstrate broad application prospects in medical fields. Carbon nanotubes are composed of inorganic materials, natural biodegradable polymer or synthetic biodegradable polymer. The composite bone tissue engineering scaffolds are constructed by particle-hole method, lyophilization, microsphere aggregation method, electrostatic spinning or three-dimensional printing. Composite scaffolds overcome the shortcomings of single material and have good biocompatibility, osteoconduction and osteoinduction. With the study of surface chemistry, toxicology, and biocompatibility, a degradable "human-friendly" carbon nanotubes composite bone tissue scaffold will be available; and under the drive of new fabrication techniques, the clinical application of carbon nanotubes composite bone tissue engineering scaffolds will be better developed. PMID:27273990

  15. Targeting obesity-related adipose tissue dysfunction to prevent cancer development and progression.

    PubMed

    Gucalp, Ayca; Iyengar, Neil M; Hudis, Clifford A; Dannenberg, Andrew J

    2016-02-01

    The incidence of obesity, a leading modifiable risk factor for common solid tumors, is increasing. Effective interventions are needed to minimize the public health implications of obesity. Although the mechanisms linking increased adiposity to malignancy are incompletely understood, growing evidence points to complex interactions among multiple systemic and tissue-specific pathways including inflamed white adipose tissue. The metabolic and inflammatory consequences of white adipose tissue dysfunction collectively provide a plausible explanation for the link between overweight/obesity and carcinogenesis. Gaining a better understanding of these underlying molecular pathways and developing risk assessment tools that identify at-risk populations will be critical in implementing effective and novel cancer prevention and management strategies.

  16. [Research progress on real-time deformable models of soft tissues for surgery simulation].

    PubMed

    Xu, Shaoping; Liu, Xiaoping; Zhang, Hua; Luo, Jie

    2010-04-01

    Biological tissues generally exhibit nonlinearity, anisotropy, quasi-incompressibility and viscoelasticity about material properties. Simulating the behaviour of elastic objects in real time is one of the current objectives of virtual surgery simulation which is still a challenge for researchers to accurately depict the behaviour of human tissues. In this paper, we present a classification of the different deformable models that have been developed. We present the advantages and disadvantages of each one. Finally, we make a comparison of deformable models and perform an evaluation of the state of the art and the future of deformable models.

  17. [The application progress of human urine derived stem cells in bone tissue engineering].

    PubMed

    Gao, Peng; Jiang, Dapeng; Li, Zhaozhu

    2016-04-01

    The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell (hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential. PMID:27029208

  18. Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation.

    PubMed

    Boué, Stéphanie; De León, Héctor; Schlage, Walter K; Peck, Michael J; Weiler, Horst; Berges, An; Vuillaume, Grégory; Martin, Florian; Friedrichs, Baerbel; Lebrun, Stefan; Meurrens, Kris; Schracke, Nadine; Moehring, Michaela; Steffen, Yvonne; Schueller, Jutta; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia

    2013-12-01

    Cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD) and a risk factor for both lung and cardiovascular (CV) diseases, which are rarely investigated concomitantly. Although smoking cessation shows clear CV risk benefit, lung-related disease risk remains higher in former smokers than in never smokers. We sought to determine the differential molecular responses of murine respiratory tissues to better understand the toxicity pathways involved in smoking-related disease risk and those related to the benefits of smoking cessation. ApoE(-/-) mice were exposed to mainstream cigarette smoke (CS) or a smoking cessation-mimicking protocol for up to 6 months and transcriptomics analysis of nasal epithelium and lung parenchyma performed. We supported our gene expression profiling approach with standard lung histopathology and bronchoalveolar lavage fluid (BALF) analysis. Many BALF analytes involved in functions ranging from inflammation to cell proliferation and tissue remodeling were found elevated in BALF. Gene expression levels of these molecules were also increased in lung tissue, suggesting that the inflammatory response was the result of local tissue activation and the contribution of recruited inflammatory cells. Gene set enrichment analysis (GSEA) of expression data from murine lungs and nasal epithelium showed distinct activation patterns of inflammation, complement, and xenobiotic metabolism pathways during CS exposure that were deactivated upon smoking cessation. Pathways involved in cell proliferation and tissue remodeling were activated by CS and progressively deactivated upon smoke exposure cessation. Differential CS-mediated responses of pulmonary and nasal tissues reflect common mechanisms but also the varying degrees of epithelial functional specialization and exposure along the respiratory tract. PMID:24096154

  19. A Cell-Regulatory Mechanism Involving Feedback between Contraction and Tissue Formation Guides Wound Healing Progression

    PubMed Central

    Valero, Clara; Javierre, Etelvina; García-Aznar, José Manuel; Gómez-Benito, María José

    2014-01-01

    Wound healing is a process driven by cells. The ability of cells to sense mechanical stimuli from the extracellular matrix that surrounds them is used to regulate the forces that cells exert on the tissue. Stresses exerted by cells play a central role in wound contraction and have been broadly modelled. Traditionally, these stresses are assumed to be dependent on variables such as the extracellular matrix and cell or collagen densities. However, we postulate that cells are able to regulate the healing process through a mechanosensing mechanism regulated by the contraction that they exert. We propose that cells adjust the contraction level to determine the tissue functions regulating all main activities, such as proliferation, differentiation and matrix production. Hence, a closed-regulatory feedback loop is proposed between contraction and tissue formation. The model consists of a system of partial differential equations that simulates the evolution of fibroblasts, myofibroblasts, collagen and a generic growth factor, as well as the deformation of the extracellular matrix. This model is able to predict the wound healing outcome without requiring the addition of phenomenological laws to describe the time-dependent contraction evolution. We have reproduced two in vivo experiments to evaluate the predictive capacity of the model, and we conclude that there is feedback between the level of cell contraction and the tissue regenerated in the wound. PMID:24681636

  20. Progress toward automatic classification of human brown adipose tissue using biomedical imaging

    NASA Astrophysics Data System (ADS)

    Gifford, Aliya; Towse, Theodore F.; Walker, Ronald C.; Avison, Malcom J.; Welch, E. B.

    2015-03-01

    Brown adipose tissue (BAT) is a small but significant tissue, which may play an important role in obesity and the pathogenesis of metabolic syndrome. Interest in studying BAT in adult humans is increasing, but in order to quantify BAT volume in a single measurement or to detect changes in BAT over the time course of a longitudinal experiment, BAT needs to first be reliably differentiated from surrounding tissue. Although the uptake of the radiotracer 18F-Fluorodeoxyglucose (18F-FDG) in adipose tissue on positron emission tomography (PET) scans following cold exposure is accepted as an indication of BAT, it is not a definitive indicator, and to date there exists no standardized method for segmenting BAT. Consequently, there is a strong need for robust automatic classification of BAT based on properties measured with biomedical imaging. In this study we begin the process of developing an automated segmentation method based on properties obtained from fat-water MRI and PET-CT scans acquired on ten healthy adult subjects.

  1. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  2. Tiling Microarray Analysis Tools

    SciTech Connect

    Nix, Davis Austin

    2005-05-04

    TiMAT is a package of 23 command line Java applications for use in the analysis of Affymetrix tiled genomic microarray data. TiMAT enables: 1) Rebuilding the genome annotation for entire tiled arrays (repeat filtering, chromosomal coordinate assignment). 2) Post processing of oligo intensity values (quantile normalization, median scaling, PMMM transformation), 3) Significance testing (Wilcoxon rank sum and signed rank tests, intensity difference and ratio tests) and Interval refinement (filtering based on multiple statistics, overlap comparisons), 4) Data visualization (detailed thumbnail/zoomed view with Interval Plots and data export to Affymetrix's Integrated Genome Browser) and Data reports (spreadsheet summaries and detailed profiles)

  3. Evolution of normal and neoplastic tissue stem cells: progress after Robert Hooke.

    PubMed

    Weissman, Irving

    2015-10-19

    The appearance of stem cells coincides with the transition from single-celled organisms to metazoans. Stem cells are capable of self-renewal as well as differentiation. Each tissue is maintained by self-renewing tissue-specific stem cells. The accumulation of mutations that lead to preleukaemia are in the blood-forming stem cell, while the transition to leukaemia stem cells occurs in the clone at a progenitor stage. All leukaemia and cancer cells escape being removed by scavenger macrophages by expressing the 'don't eat me' signal CD47. Blocking antibodies to CD47 are therapeutics for all cancers, and are currently being tested in clinical trials in the US and UK. PMID:26416675

  4. Evolution of normal and neoplastic tissue stem cells: progress after Robert Hooke.

    PubMed

    Weissman, Irving

    2015-10-19

    The appearance of stem cells coincides with the transition from single-celled organisms to metazoans. Stem cells are capable of self-renewal as well as differentiation. Each tissue is maintained by self-renewing tissue-specific stem cells. The accumulation of mutations that lead to preleukaemia are in the blood-forming stem cell, while the transition to leukaemia stem cells occurs in the clone at a progenitor stage. All leukaemia and cancer cells escape being removed by scavenger macrophages by expressing the 'don't eat me' signal CD47. Blocking antibodies to CD47 are therapeutics for all cancers, and are currently being tested in clinical trials in the US and UK.

  5. Evolution of normal and neoplastic tissue stem cells: progress after Robert Hooke

    PubMed Central

    Weissman, Irving

    2015-01-01

    The appearance of stem cells coincides with the transition from single-celled organisms to metazoans. Stem cells are capable of self-renewal as well as differentiation. Each tissue is maintained by self-renewing tissue-specific stem cells. The accumulation of mutations that lead to preleukaemia are in the blood-forming stem cell, while the transition to leukaemia stem cells occurs in the clone at a progenitor stage. All leukaemia and cancer cells escape being removed by scavenger macrophages by expressing the 'don't eat me' signal CD47. Blocking antibodies to CD47 are therapeutics for all cancers, and are currently being tested in clinical trials in the US and UK. PMID:26416675

  6. LDRD Progress Report: Radioimmunotherapy using oxide nanoparticles: Radionuclide contaiment and mitigation of normal tissue toxicity.

    SciTech Connect

    Rondinone, Adam Justin; Dai, Sheng; Mirzadeh, Saed; Kennel, Steve J

    2005-10-01

    Radionuclides with specific emission properties can be incorporated into metal-chalcogenide and metal-oxide nanoparticles. Coupled to antibodies, these conjugates could be injected into the bloodstream to target and destroy non-solid tumors or target organs for radioimaging. In the first year of this project, two types of radioactive nanoparticles, CdTe: {sup 125m}Te and Y{sub 2}O{sub 3}: {sup 170}Tm were synthesized and coupled to antibodies specific to murine epithelial lung tissue. The nanoparticles successfully target the lung tissue in vivo. Some leaching of the radioisotope was observed. The coming year will explore other types of nanoparticles (other crystal chemistries) in order to minimize leaching.

  7. Ecotoxicogenomics: Microarray interlaboratory comparability.

    PubMed

    Vidal-Dorsch, Doris E; Bay, Steven M; Moore, Shelly; Layton, Blythe; Mehinto, Alvine C; Vulpe, Chris D; Brown-Augustine, Marianna; Loguinov, Alex; Poynton, Helen; Garcia-Reyero, Natàlia; Perkins, Edward J; Escalon, Lynn; Denslow, Nancy D; Cristina, Colli-Dula R; Doan, Tri; Shukradas, Shweta; Bruno, Joy; Brown, Lorraine; Van Agglen, Graham; Jackman, Paula; Bauer, Megan

    2016-02-01

    Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training.

  8. LCM assisted biomarker discovery from archival neoplastic gastrointestinal tissues.

    PubMed

    Meitner, Patricia A; Resnick, Murray B

    2011-01-01

    Expression array analysis of epithelial mRNA to identify biomarkers of premalignant and malignant conditions in the gastrointestinal (GI) tract is an area of intense study. Archived formalin-fixed paraffin-embedded (FFPE) tissues documenting these changes are readily available and should be a valuable resource for retrospective analysis. Laser capture microdissection of defined areas of epithelial cells at different stages of neoplastic progression is described together with methods for prequalification of RNA in FFPE tissue blocks selected for analysis. Paradise reagents specifically designed for isolation and amplification of RNA from FFPE archival tissue specimens are used to prepare probes for the human X3P microarray from Affymetrix.

  9. Effect of growth differentiation factor-9 (GDF-9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues.

    PubMed

    Abdel-Ghani, M A; El-Sherry, T M; Abdelhafeez, H H

    2016-10-01

    To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen-thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to

  10. Effect of growth differentiation factor-9 (GDF-9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues.

    PubMed

    Abdel-Ghani, M A; El-Sherry, T M; Abdelhafeez, H H

    2016-10-01

    To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen-thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to

  11. JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection

    PubMed Central

    Wharton, Keith A.; Quigley, Catherine; Themeles, Marian; Dunstan, Robert W.; Doyle, Kathryn; Cahir-McFarland, Ellen; Wei, Jing; Buko, Alex; Reid, Carl E.; Sun, Chao; Carmillo, Paul; Sur, Gargi; Carulli, John P.; Mansfield, Keith G.; Westmoreland, Susan V.; Staugaitis, Susan M.; Fox, Robert J.; Meier, Werner; Goelz, Susan E.

    2016-01-01

    Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath. PMID:27191595

  12. Progress in the tissue engineering and stem cell industry "are we there yet?".

    PubMed

    Jaklenec, Ana; Stamp, Andrea; Deweerd, Elizabeth; Sherwin, Angela; Langer, Robert

    2012-06-01

    This report presents a detailed update to our 2008 publication on the tissue engineering (TE) and stem cell industry. Data are reported through mid 2011 showing an almost three-fold growth in commercial sales over the past 4 years. In addition, the number of companies selling products or offering services has increased over two-fold to 106, and they are generating a remarkable $3.5 billion in sales. Overall, the TE and stem cell sector is spending $3.6 billion and employing almost 14,000 employees. These data suggest the TE and stem cell industry has stabilized and is on a path pointing toward continued success.

  13. Harnessing the power of gene microarrays for the study of brain aging and Alzheimer's disease: statistical reliability and functional correlation.

    PubMed

    Blalock, E M; Chen, K-C; Stromberg, A J; Norris, C M; Kadish, I; Kraner, S D; Porter, N M; Landfield, P W

    2005-11-01

    During normal brain aging, numerous alterations develop in the physiology, biochemistry and structure of neurons and glia. Aging changes occur in most brain regions and, in the hippocampus, have been linked to declining cognitive performance in both humans and animals. Age-related changes in hippocampal regions also may be harbingers of more severe decrements to come from neurodegenerative disorders such as Alzheimer's disease (AD). However, unraveling the mechanisms underlying brain aging, AD and impaired function has been difficult because of the complexity of the networks that drive these aging-related changes. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important new research tool that potentially can provide the investigative power needed to address the complexity of brain aging/neurodegenerative processes. However, along with this new analytic power, microarrays bring several major bioinformatics and resource problems that frequently hinder the optimal application of this technology. In particular, microarray analyses generate extremely large and unwieldy data sets and are subject to high false positive and false negative rates. Concerns also have been raised regarding their accuracy and uniformity. Furthermore, microarray analyses can result in long lists of altered genes, most of which may be difficult to evaluate for functional relevance. These and other problems have led to some skepticism regarding the reliability and functional usefulness of microarray data and to a general view that microarray data should be validated by an independent method. Given recent progress, however, we suggest that the major problem for current microarray research is no longer validity of expression measurements, but rather, the reliability of inferences from the data, an issue more appropriately redressed by statistical approaches than by validation with a separate method. If tested using statistically

  14. Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays*

    PubMed Central

    Babel, Ingrid; Barderas, Rodrigo; Díaz-Uriarte, Ramón; Martínez-Torrecuadrada, Jorge Luis; Sánchez-Carbayo, Marta; Casal, J. Ignacio

    2009-01-01

    There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. PMID:19638618

  15. The Genopolis Microarray Database

    PubMed Central

    Splendiani, Andrea; Brandizi, Marco; Even, Gael; Beretta, Ottavio; Pavelka, Norman; Pelizzola, Mattia; Mayhaus, Manuel; Foti, Maria; Mauri, Giancarlo; Ricciardi-Castagnoli, Paola

    2007-01-01

    Background Gene expression databases are key resources for microarray data management and analysis and the importance of a proper annotation of their content is well understood. Public repositories as well as microarray database systems that can be implemented by single laboratories exist. However, there is not yet a tool that can easily support a collaborative environment where different users with different rights of access to data can interact to define a common highly coherent content. The scope of the Genopolis database is to provide a resource that allows different groups performing microarray experiments related to a common subject to create a common coherent knowledge base and to analyse it. The Genopolis database has been implemented as a dedicated system for the scientific community studying dendritic and macrophage cells functions and host-parasite interactions. Results The Genopolis Database system allows the community to build an object based MIAME compliant annotation of their experiments and to store images, raw and processed data from the Affymetrix GeneChip® platform. It supports dynamical definition of controlled vocabularies and provides automated and supervised steps to control the coherence of data and annotations. It allows a precise control of the visibility of the database content to different sub groups in the community and facilitates exports of its content to public repositories. It provides an interactive users interface for data analysis: this allows users to visualize data matrices based on functional lists and sample characterization, and to navigate to other data matrices defined by similarity of expression values as well as functional characterizations of genes involved. A collaborative environment is also provided for the definition and sharing of functional annotation by users. Conclusion The Genopolis Database supports a community in building a common coherent knowledge base and analyse it. This fills a gap between a local

  16. Estrogen receptor (ER)α-regulated lipocalin 2 expression in adipose tissue links obesity with breast cancer progression.

    PubMed

    Drew, Brian G; Hamidi, Habib; Zhou, Zhenqi; Villanueva, Claudio J; Krum, Susan A; Calkin, Anna C; Parks, Brian W; Ribas, Vicent; Kalajian, Nareg Y; Phun, Jennifer; Daraei, Pedram; Christofk, Heather R; Hewitt, Sylvia C; Korach, Kenneth S; Tontonoz, Peter; Lusis, Aldons J; Slamon, Dennis J; Hurvitz, Sara A; Hevener, Andrea L

    2015-02-27

    Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity.

  17. ChIP-seq in steatohepatitis and normal liver tissue identifies candidate disease mechanisms related to progression to cancer

    PubMed Central

    2013-01-01

    Background Steatohepatitis occurs in alcoholic liver disease and may progress to liver cirrhosis and hepatocellular carcinoma. Its molecular pathogenesis is to a large degree unknown. Histone modifications play a key role in transcriptional regulations as marks for silencing and activation of gene expression and as marks for functional elements. Many transcription factors (TFs) are crucial for the control of the genes involved in metabolism, and abnormality in their function may lead to disease. Methods We performed ChIP-seq of the histone modifications H3K4me1, H3K4me3 and H3K27ac and a candidate transcription factor (USF1) in liver tissue from patients with steatohepatitis and normal livers and correlated results to mRNA-expression and genotypes. Results We found several regions that are differentially enriched for histone modifications between disease and normal tissue, and qRT-PCR results indicated that the expression of the tested genes strongly correlated with differential enrichment of histone modifications but is independent of USF1 enrichment. By gene ontology analysis of differentially modified genes we found many disease associated genes, some of which had previously been implicated in the etiology of steatohepatitis. Importantly, the genes associated to the strongest histone peaks in the patient were over-represented in cancer specific pathways suggesting that the tissue was on a path to develop to cancer, a common complication to the disease. We also found several novel SNPs and GWAS catalogue SNPs that are candidates to be functional and therefore needs further study. Conclusion In summary we find that analysis of chromatin features in tissue samples provides insight into disease mechanisms. PMID:24206787

  18. Estrogen Receptor (ER)α-regulated Lipocalin 2 Expression in Adipose Tissue Links Obesity with Breast Cancer Progression*

    PubMed Central

    Drew, Brian G.; Hamidi, Habib; Zhou, Zhenqi; Villanueva, Claudio J.; Krum, Susan A.; Calkin, Anna C.; Parks, Brian W.; Ribas, Vicent; Kalajian, Nareg Y.; Phun, Jennifer; Daraei, Pedram; Christofk, Heather R.; Hewitt, Sylvia C.; Korach, Kenneth S.; Tontonoz, Peter; Lusis, Aldons J.; Slamon, Dennis J.; Hurvitz, Sara A.; Hevener, Andrea L.

    2015-01-01

    Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity. PMID:25468909

  19. DNA microarray application in ecotoxicology: experimental design, microarray scanning, and factors affecting transcriptional profiles in a small fish species.

    PubMed

    Wang, Rong-Lin; Biales, Adam; Bencic, David; Lattier, David; Kostich, Mitch; Villeneuve, Dan; Ankley, Gerald T; Lazorchak, Jim; Toth, Greg

    2008-03-01

    The research presented here is part of a larger study of the molecular mode of action of endocrine-disrupting chemicals targeting the hypothalamic-pituitary-gonadal axis in zebrafish (Danio rerio). It addresses several issues critical to microarray application in aquatic ecotoxicology: experimental design, microarray scanning, gene expression intensity distribution, and the effect of experimental parameters on the zebrafish transcriptome. Expression profiles from various tissues of individual zebrafish exposed to 17alpha-ethinylestradiol (30 ng/L), fadrozole (25 micro.g/L), or 17beta-trenbolone (3.0 microg/L) for 48 or 96 h were examined with the Agilent Oligo Microarray (G2518A). As a flexible and efficient alternative to the designs commonly used in microarray studies, an unbalanced incomplete block design was found to be well suited for this work, as evidenced by high data reproducibility, low microarray-to-microarray variability, and little gene-specific dye bias. Random scanner noise had little effect on data reproducibility. A low-level, slightly variable Cyanine 3 (Cy3) contaminant was revealed by hyperspectral imaging, suggesting fluorescence contamination as a potential contributor to the large variance associated with weakly expressed genes. Expression intensities of zebrafish genes were skewed toward the lower end of their distribution range, and more weakly expressed genes tended to have larger variances. Tissue type, followed in descending order by gender, chemical treatment, and exposure duration, had the greatest effect on the overall gene expression profiles, a finding potentially critical to experimental design optimization. Overall, congruence was excellent between quantitative polymerase chain reaction results and microarray profiles of 13 genes examined across a subset of 20 pairs of ovarian samples. These findings will help to improve applications of microarrays in future ecotoxicological studies.

  20. Living-Cell Microarrays

    PubMed Central

    Yarmush, Martin L.; King, Kevin R.

    2011-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

  1. Tiling Microarray Analysis Tools

    2005-05-04

    TiMAT is a package of 23 command line Java applications for use in the analysis of Affymetrix tiled genomic microarray data. TiMAT enables: 1) Rebuilding the genome annotation for entire tiled arrays (repeat filtering, chromosomal coordinate assignment). 2) Post processing of oligo intensity values (quantile normalization, median scaling, PMMM transformation), 3) Significance testing (Wilcoxon rank sum and signed rank tests, intensity difference and ratio tests) and Interval refinement (filtering based on multiple statistics, overlap comparisons),more » 4) Data visualization (detailed thumbnail/zoomed view with Interval Plots and data export to Affymetrix's Integrated Genome Browser) and Data reports (spreadsheet summaries and detailed profiles)« less

  2. Gene delivery in bone tissue engineering: progress and prospects using viral and nonviral strategies.

    PubMed

    Partridge, Kris A; Oreffo, Richard O C

    2004-01-01

    Bone tissue loss as a consequence of the natural aging process or as a result of trauma and degenerative disease has led to the need for procedures to generate cartilage and bone for a variety of orthopedic applications. The ability to transfer genes into multipotential mesenchymal stem cells, while still in its infancy, offers considerable therapeutic hope in a variety of musculoskeletal disorders. However, the choice of gene delivery method is key. This review examines the various techniques and methods currently available to enable gene transfer into a target population from viral methods (transduction) to nonviral (transfection) methods and the limitations associated with each method. The potential applications and current understanding of each method are presented. Given the demographic challenge of an aging population, the ultimate goal remains the development of simple, safe, and reproducible strategies for gene delivery that will address the pressing orthopedic clinical imperatives of many.

  3. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

    PubMed Central

    Miller, Melissa B.; Tang, Yi-Wei

    2009-01-01

    Summary: The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles. PMID:19822891

  4. Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning

    PubMed Central

    Hadjivasiliou, Zena; Bonin, Hope; He, Li; Perrimon, Norbert; Charras, Guillaume; Baum, Buzz

    2016-01-01

    Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning. PMID:27226324

  5. Microarray platform for omics analysis

    NASA Astrophysics Data System (ADS)

    Mecklenburg, Michael; Xie, Bin

    2001-09-01

    Microarray technology has revolutionized genetic analysis. However, limitations in genome analysis has lead to renewed interest in establishing 'omic' strategies. As we enter the post-genomic era, new microarray technologies are needed to address these new classes of 'omic' targets, such as proteins, as well as lipids and carbohydrates. We have developed a microarray platform that combines self- assembling monolayers with the biotin-streptavidin system to provide a robust, versatile immobilization scheme. A hydrophobic film is patterned on the surface creating an array of tension wells that eliminates evaporation effects thereby reducing the shear stress to which biomolecules are exposed to during immobilization. The streptavidin linker layer makes it possible to adapt and/or develop microarray based assays using virtually any class of biomolecules including: carbohydrates, peptides, antibodies, receptors, as well as them ore traditional DNA based arrays. Our microarray technology is designed to furnish seamless compatibility across the various 'omic' platforms by providing a common blueprint for fabricating and analyzing arrays. The prototype microarray uses a microscope slide footprint patterned with 2 by 96 flat wells. Data on the microarray platform will be presented.

  6. Sporophytic ovule tissues modulate the initiation and progression of apomixis in Hieracium.

    PubMed

    Tucker, Matthew R; Okada, Takashi; Johnson, Susan D; Takaiwa, Fumio; Koltunow, Anna M G

    2012-05-01

    Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.

  7. Protein microarray: sensitive and effective immunodetection for drug residues

    PubMed Central

    2010-01-01

    Background Veterinary drugs such as clenbuterol (CL) and sulfamethazine (SM2) are low molecular weight (<1000 Da) compounds, or haptens, that are difficult to develop immunoassays due to their low immunogenicity. In this study, we conjugated the drugs to ovalbumin to increase their immunogenicity for antiserum production in rabbits and developed a protein microarray immunoassay for detection of clenbuterol and sulfamethazine. The sensitivity of this approach was then compared to traditional ELISA technique. Results The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA) showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g) than the ci-ELISA (0.1 ng/g) for detection of CL residues. Conclusions The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique. PMID:20158905

  8. Microarray Analysis of Microbial Weathering

    NASA Astrophysics Data System (ADS)

    Olsson-Francis, K.; van Houdt, R.; Leys, N.; Mergeay, M.; Cockell, C. S.

    2010-04-01

    Microarray analysis of the heavy metal resistant bacterium, Cupriavidus metallidurans CH34 was used to investigate the genes involved in the weathering. The results demonstrated that large porin and membrane transporter genes were unregulated.

  9. Are glycan biosensors an alternative to glycan microarrays?

    PubMed Central

    Hushegyi, A.

    2016-01-01

    Complex carbohydrates (glycans) play an important role in nature and study of their interaction with proteins or intact cells can be useful for understanding many physiological and pathological processes. Such interactions have been successfully interrogated in a highly parallel way using glycan microarrays, but this technique has some limitations. Thus, in recent years glycan biosensors in numerous progressive configurations have been developed offering distinct advantages compared to glycan microarrays. Thus, in this review advances achieved in the field of label-free glycan biosensors are discussed. PMID:27231487

  10. A lectin-based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile.

    PubMed

    Accogli, Gianluca; Desantis, Salvatore; Martino, Nicola Antonio; Dell'Aquila, Maria Elena; Gemeiner, Peter; Katrlík, Jaroslav

    2016-10-01

    The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.

  11. Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

    PubMed

    Sjöberg, Ronald; Mattsson, Cecilia; Andersson, Eni; Hellström, Cecilia; Uhlen, Mathias; Schwenk, Jochen M; Ayoglu, Burcu; Nilsson, Peter

    2016-09-25

    High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions. PMID:26417875

  12. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  13. Microarray analysis of the developing cortex.

    PubMed

    Semeralul, Mawahib O; Boutros, Paul C; Likhodi, Olga; Okey, Allan B; Van Tol, Hubert H M; Wong, Albert H C

    2006-12-01

    Abnormal development of the prefrontal cortex (PFC) is associated with a number of neuropsychiatric disorders that have an onset in childhood or adolescence. Although the basic laminar structure of the PFC is established in utero, extensive remodeling continues into adolescence. To map the overall pattern of changes in cortical gene transcripts during postnatal development, we made serial measurements of mRNA levels in mouse PFC using oligonucleotide microarrays. We observed changes in mRNA transcripts consistent with known postnatal morphological and biochemical events. Overall, most transcripts that changed significantly showed a progressive decrease in abundance after birth, with the majority of change between postnatal weeks 2 and 4. Genes with cell proliferative, cytoskeletal, extracellular matrix, plasma membrane lipid/transport, protein folding, and regulatory functions had decreases in mRNA levels. Quantitative PCR verified the microarray results for six selected genes: DNA methyltransferase 3A (Dnmt3a), procollagen, type III, alpha 1 (Col3a1), solute carrier family 16 (monocarboxylic acid transporters), member 1 (Slc16a1), MARCKS-like 1 (Marcksl1), nidogen 1 (Nid1) and 3-hydroxybutyrate dehydrogenase (heart, mitochondrial) (Bdh).

  14. Protein microarrays: prospects and problems.

    PubMed

    Kodadek, T

    2001-02-01

    Protein microarrays are potentially powerful tools in biochemistry and molecular biology. Two types of protein microarrays are defined. One, termed a protein function array, will consist of thousands of native proteins immobilized in a defined pattern. Such arrays can be utilized for massively parallel testing of protein function, hence the name. The other type is termed a protein-detecting array. This will consist of large numbers of arrayed protein-binding agents. These arrays will allow for expression profiling to be done at the protein level. In this article, some of the major technological challenges to the development of protein arrays are discussed, along with potential solutions.

  15. Progressive natural killer cell dysfunction associated with alterations in subset proportions and receptor expression in soft-tissue sarcoma patients.

    PubMed

    Bücklein, Veit; Adunka, Tina; Mendler, Anna N; Issels, Rolf; Subklewe, Marion; Schmollinger, Jan C; Noessner, Elfriede

    2016-07-01

    Immunotherapy is currently investigated as treatment option in many types of cancer. So far, results from clinical trials have demonstrated that significant benefit from immunomodulatory therapies is restricted to patients with select histologies. To broaden the potential use of these therapies, a deeper understanding for mechanisms of immunosuppression in patients with cancer is needed. Soft-tissue sarcoma (STS) presents a medical challenge with significant mortality even after multimodal treatment. We investigated function and immunophenotype of peripheral natural killer (NK) cells from chemotherapy-naive STS patients (1st line) and STS patients with progression or relapse after previous chemotherapeutic treatment (2nd line). We found NK cells from peripheral blood of both STS patient cohorts to be dysfunctional, being unable to lyse K562 target cells while NK cells from renal cell cancer (RCC) patients did not display attenuated lytic activity. Ex vivo stimulation of NK cells from STS patients with interleukin-2 plus TKD restored cytotoxic function. Furthermore, altered NK cell subset composition with reduced proportions of CD56(dim) cells could be demonstrated, increasing from 1st- to 2nd-line patients. 2nd-line patients additionally displayed significantly reduced expression of receptors (NKG2D), mediators (CD3ζ), and effectors (perforin) of NK cell activation. In these patients, we also detected fewer NK cells with CD57 expression, a marker for terminally differentiated cytotoxic NK cells. Our results elucidate mechanisms of NK cell dysfunction in STS patients with advanced disease. Markers like NKG2D, CD3ζ, and perforin are candidates to characterize NK cells with effective antitumor function for immunotherapeutic interventions. PMID:27622032

  16. Progressive natural killer cell dysfunction associated with alterations in subset proportions and receptor expression in soft-tissue sarcoma patients

    PubMed Central

    Bücklein, Veit; Adunka, Tina; Mendler, Anna N.; Issels, Rolf; Subklewe, Marion; Schmollinger, Jan C.; Noessner, Elfriede

    2016-01-01

    ABSTRACT Immunotherapy is currently investigated as treatment option in many types of cancer. So far, results from clinical trials have demonstrated that significant benefit from immunomodulatory therapies is restricted to patients with select histologies. To broaden the potential use of these therapies, a deeper understanding for mechanisms of immunosuppression in patients with cancer is needed. Soft-tissue sarcoma (STS) presents a medical challenge with significant mortality even after multimodal treatment. We investigated function and immunophenotype of peripheral natural killer (NK) cells from chemotherapy-naive STS patients (1st line) and STS patients with progression or relapse after previous chemotherapeutic treatment (2nd line). We found NK cells from peripheral blood of both STS patient cohorts to be dysfunctional, being unable to lyse K562 target cells while NK cells from renal cell cancer (RCC) patients did not display attenuated lytic activity. Ex vivo stimulation of NK cells from STS patients with interleukin-2 plus TKD restored cytotoxic function. Furthermore, altered NK cell subset composition with reduced proportions of CD56dim cells could be demonstrated, increasing from 1st- to 2nd-line patients. 2nd-line patients additionally displayed significantly reduced expression of receptors (NKG2D), mediators (CD3ζ), and effectors (perforin) of NK cell activation. In these patients, we also detected fewer NK cells with CD57 expression, a marker for terminally differentiated cytotoxic NK cells. Our results elucidate mechanisms of NK cell dysfunction in STS patients with advanced disease. Markers like NKG2D, CD3ζ, and perforin are candidates to characterize NK cells with effective antitumor function for immunotherapeutic interventions. PMID:27622032

  17. Microarray Developed on Plastic Substrates.

    PubMed

    Bañuls, María-José; Morais, Sergi B; Tortajada-Genaro, Luis A; Maquieira, Ángel

    2016-01-01

    There is a huge potential interest to use synthetic polymers as versatile solid supports for analytical microarraying. Chemical modification of polycarbonate (PC) for covalent immobilization of probes, micro-printing of protein or nucleic acid probes, development of indirect immunoassay, and development of hybridization protocols are described and discussed. PMID:26614067

  18. Microfluidic microarray systems and methods thereof

    SciTech Connect

    West, Jay A. A.; Hukari, Kyle W.; Hux, Gary A.

    2009-04-28

    Disclosed are systems that include a manifold in fluid communication with a microfluidic chip having a microarray, an illuminator, and a detector in optical communication with the microarray. Methods for using these systems for biological detection are also disclosed.

  19. The Microarray Revolution: Perspectives from Educators

    ERIC Educational Resources Information Center

    Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

    2004-01-01

    In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research,…

  20. Current Protocols in Chemical Biology Construction and Use of Glycan Microarrays

    PubMed Central

    Campbell, Christopher T.; Zhang, Yalong; Gildersleeve, Jeffrey C.

    2011-01-01

    Glycosylation is an important post-translational modification that influences many biological processes critical for development, normal physiologic function, and diseases. Unfortunately, progress towards understanding the roles of glycans in biology has been slow due to the challenges of studying glycans and the proteins that interact with them. Glycan microarrays provide a high-throughput approach for the rapid analysis of carbohydrate-macromolecule interactions. Protocols detailed here are intended to help laboratories with basic familiarity of DNA or protein microarrays to begin printing and performing assays using glycan microarrays. Basic and advanced data processing are also detailed, along with strategies for improving reproducibility of data collected with glycan arrays. PMID:23836542

  1. Disc-based microarrays: principles and analytical applications.

    PubMed

    Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2016-07-01

    The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays. PMID:26922341

  2. Microarray technology for use in molecular epidemiology.

    PubMed

    Vernon, Suzanne D; Whistler, Toni

    2007-01-01

    Microarrays are a powerful laboratory tool for the simultaneous assessment of the activity of thousands genes. Remarkable advances in biological sample collection, preparation and automation of hybridization have enabled the application of microarray technology to large, population-based studies. Now, microarrays have the potential to serve as screening tools for the detection of altered gene expression activity that might contribute to diseases in human populations. Reproducible and reliable microarray results depend on multiple factors. In this chapter, biological sample parameters are introduced that should be considered for any microarray experiment. Then, the microarray technology that we have successfully applied to limited biological sample from all our molecular epidemiology studies is detailed. This reproducible and reliable approach for using microarrays should be applicable to any biological questions asked.

  3. Phenotypic MicroRNA Microarrays

    PubMed Central

    Kwon, Yong-Jun; Heo, Jin Yeong; Kim, Hi Chul; Kim, Jin Yeop; Liuzzi, Michel; Soloveva, Veronica

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

  4. Self-Assembling Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ramachandran, Niroshan; Hainsworth, Eugenie; Bhullar, Bhupinder; Eisenstein, Samuel; Rosen, Benjamin; Lau, Albert Y.; C. Walter, Johannes; LaBaer, Joshua

    2004-07-01

    Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

  5. 5α-Reductase Type 3 Expression in Human Benign and Malignant Tissues: A Comparative Analysis During Prostate Cancer Progression

    PubMed Central

    Godoy, Alejandro; Kawinski, Elzbieta; Li, Yun; Oka, Daizo; Alexiev, Borislav; Azzouni, Faris; Titus, Mark A.; Mohler, James L.

    2015-01-01

    BACKGROUND A third isozyme of human 5α-steroid reductase, 5α-reductase-3, was identified in prostate tissue at the mRNA level. However, the levels of 5α-reductase-3 protein expression and its cellular localization in human tissues remain unknown. METHODS A specific monoclonal antibody was developed, validated, and used to characterize for the first time the expression of 5α-reductase-3 protein in 18 benign and 26 malignant human tissue types using immunostaining analyses. RESULTS AND CONCLUSIONS In benign tissues, 5α-reductase-3 immunostaining was high in conventional androgen-regulated human tissues, such as skeletal muscle and prostate. However, high levels of expression also were observed in non-conventional androgen-regulated tissues, which suggest either multiples target tissues for androgens or different functions of 5α-reductase-3 among human tissues. In malignant tissues, 5α-reductase-3 immunostaining was ubiquitous but particularly over-expressed in some cancers compared to their benign counterparts, which suggests a potential role for 5α-reductase-3 as a biomarker of malignancy. In benign prostate, 5α-reductase-3 immunostaining was localized to basal epithelial cells, with no immunostaining observed in secretory/luminal epithelial cells. In high-grade prostatic intraepithelial neoplasia (HGPIN), 5α-reductase-3 immunostaining was localized in both basal epithelial cells and neoplastic epithelial cells characteristic of HGPIN. In androgen-stimulated and castration-recurrent prostate cancer (CaP), 5α-reductase-3 immunostaining was present in most epithelial cells and at similar levels, and at levels higher than observed in benign prostate. Analyses of expression and functionality of 5α-reductase-3 in human tissues may prove useful for development of treatment for benign prostatic enlargement and prevention and treatment of CaP. PMID:21557268

  6. Assessing Agreement between miRNA Microarray Platforms

    PubMed Central

    Bassani, Niccolò P.; Ambrogi, Federico; Biganzoli, Elia M.

    2014-01-01

    Over the last few years, miRNA microarray platforms have provided great insights into the biological mechanisms underlying the onset and development of several diseases. However, only a few studies have evaluated the concordance between different microarray platforms using methods that took into account measurement error in the data. In this work, we propose the use of a modified version of the Bland–Altman plot to assess agreement between microarray platforms. To this aim, two samples, one renal tumor cell line and a pool of 20 different human normal tissues, were profiled using three different miRNA platforms (Affymetrix, Agilent, Illumina) on triplicate arrays. Intra-platform reliability was assessed by calculating pair-wise concordance correlation coefficients (CCC) between technical replicates and overall concordance correlation coefficient (OCCC) with bootstrap percentile confidence intervals, which revealed moderate-to-good repeatability of all platforms for both samples. Modified Bland–Altman analysis revealed good patterns of concordance for Agilent and Illumina, whereas Affymetrix showed poor-to-moderate agreement for both samples considered. The proposed method is useful to assess agreement between array platforms by modifying the original Bland–Altman plot to let it account for measurement error and bias correction and can be used to assess patterns of concordance between other kinds of arrays other than miRNA microarrays.

  7. Analysis of microarray experiments of gene expression profiling

    PubMed Central

    Tarca, Adi L.; Romero, Roberto; Draghici, Sorin

    2008-01-01

    The study of gene expression profiling of cells and tissue has become a major tool for discovery in medicine. Microarray experiments allow description of genome-wide expression changes in health and disease. The results of such experiments are expected to change the methods employed in the diagnosis and prognosis of disease in obstetrics and gynecology. Moreover, an unbiased and systematic study of gene expression profiling should allow the establishment of a new taxonomy of disease for obstetric and gynecologic syndromes. Thus, a new era is emerging in which reproductive processes and disorders could be characterized using molecular tools and fingerprinting. The design, analysis, and interpretation of microarray experiments require specialized knowledge that is not part of the standard curriculum of our discipline. This article describes the types of studies that can be conducted with microarray experiments (class comparison, class prediction, class discovery). We discuss key issues pertaining to experimental design, data preprocessing, and gene selection methods. Common types of data representation are illustrated. Potential pitfalls in the interpretation of microarray experiments, as well as the strengths and limitations of this technology, are highlighted. This article is intended to assist clinicians in appraising the quality of the scientific evidence now reported in the obstetric and gynecologic literature. PMID:16890548

  8. Lectin microarrays for glycomic analysis.

    PubMed

    Gupta, Garima; Surolia, Avadhesha; Sampathkumar, Srinivasa-Gopalan

    2010-08-01

    Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. Glycocalyx of mammalian cells actively participate in cell-cell, cell-matrix, and cell-pathogen interactions, which impact embryogenesis, growth and development, homeostasis, infection and immunity, signaling, malignancy, and metabolic disorders. Relative to genomics and proteomics, glycomics is just growing out of infancy with great potential in biomedicine for biomarker discovery, diagnosis, and treatment. However, the immense diversity and complexity of glycan structures and their multiple modes of interactions with proteins pose great challenges for development of analytical tools for delineating structure function relationships and understanding glyco-code. Several tools are being developed for glycan profiling based on chromatography, mass spectrometry, glycan microarrays, and glyco-informatics. Lectins, which have long been used in glyco-immunology, printed on a microarray provide a versatile platform for rapid high throughput analysis of glycoforms of biological samples. Herein, we summarize technological advances in lectin microarrays and critically review their impact on glycomics analysis. Challenges remain in terms of expansion to include nonplant derived lectins, standardization for routine clinical use, development of recombinant lectins, and exploration of plant kingdom for discovery of novel lectins. PMID:20726799

  9. Lectin microarrays for glycomic analysis.

    PubMed

    Gupta, Garima; Surolia, Avadhesha; Sampathkumar, Srinivasa-Gopalan

    2010-08-01

    Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. Glycocalyx of mammalian cells actively participate in cell-cell, cell-matrix, and cell-pathogen interactions, which impact embryogenesis, growth and development, homeostasis, infection and immunity, signaling, malignancy, and metabolic disorders. Relative to genomics and proteomics, glycomics is just growing out of infancy with great potential in biomedicine for biomarker discovery, diagnosis, and treatment. However, the immense diversity and complexity of glycan structures and their multiple modes of interactions with proteins pose great challenges for development of analytical tools for delineating structure function relationships and understanding glyco-code. Several tools are being developed for glycan profiling based on chromatography, mass spectrometry, glycan microarrays, and glyco-informatics. Lectins, which have long been used in glyco-immunology, printed on a microarray provide a versatile platform for rapid high throughput analysis of glycoforms of biological samples. Herein, we summarize technological advances in lectin microarrays and critically review their impact on glycomics analysis. Challenges remain in terms of expansion to include nonplant derived lectins, standardization for routine clinical use, development of recombinant lectins, and exploration of plant kingdom for discovery of novel lectins.

  10. Three-dimensional lithographically-defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression

    SciTech Connect

    Nelson, Celeste M.; Inman, Jamie L.; Bissell, Mina J.

    2008-02-13

    Here we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen gels. Epithelial cells are seeded into the cavities and covered with a second layer of collagen. The cells reorganize into hollow tissues corresponding to the geometry of the cavities. Patterned tissue arrays can be produced in 3-4 h and will undergo morphogenesis over the following one to three days. The protocol can easily be adapted to study a variety of tissues and aspects of normal and neoplastic development.

  11. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  12. Components of the endocannabinoid and dopamine systems are dysregulated in Huntington's disease: analysis of publicly available microarray datasets

    PubMed Central

    Laprairie, Robert B; Bagher, Amina M; Precious, Sophie V; Denovan-Wright, Eileen M

    2015-01-01

    The endocannabinoid system (ECS) and the dopaminergic system (DAS) are two major regulators of basal ganglia function. During Huntington's disease (HD) pathogenesis, the expression of genes in both the ECS and DAS is dysregulated. The purpose of this study was to determine the changes that were consistently observed in the ECS and DAS during HD progression in the central nervous system (CNS) and in the periphery in different models of HD and human HD tissue. To do this, we conducted a meta-analysis of differential gene expression in the ECS and DAS using publicly available microarray data. The consolidated data were summarized as observed changes in gene expression (OCGE) using a weighted sum for each gene. In addition, consolidated data were compared to previously published studies that were not available in the gene expression omnibus (GEO) database. The resulting data confirm gene expression changes observed using different approaches and provide novel insights into the consistency between changes observed in human tissue and various models, as well as disease stage- and tissue-specific transcriptional dysregulation in HD. The major implication of the systems-wide data presented here is that therapeutic strategies targeting the ECS or DAS must consider the dynamic changes in gene expression over time and in different body areas, which occur during HD progression and the interconnectedness of the two systems. PMID:25692022

  13. Immunohistochemistry Study of P53 and C-erbB-2 Expression in Trophoblastic Tissue and Their Predictive Values in Diagnosing Malignant Progression of Simple Molar Pregnancy

    PubMed Central

    Hasanzadeh, Malihe; Sharifi, Norrie; Farazestanian, Marjaneh; Nazemian, Seyed Saman; Madani Sani, Faezeh

    2016-01-01

    Background Finding a tumor marker to predict the aggressive behavior of molar pregnancy in early stages has yet been a topic for studies. Objectives In this survey we planned to study patients with molar pregnancy to 1) assess the p53 and c-erbB-2 expression in trophoblastic tissue, 2) to study the relationship between their expression intensity and progression of a molar pregnancy to gestational trophoblastic neoplasia, and 3) to determine a cut off value for the amount of p53 and c-erbB-2 expression which might correlate with aggressive behavior of molar pregnancy. Patients and Methods In a prospective cross sectional study by using a high accuracy technique EnVision Tm system for immunohistochemistry staining of molar pregnancy samples, we evaluated p53 and c-erbB-2 expression in cytotrophoblast and syncytiotrophoblast and the correlation of their expression with progression of molar pregnancy to gestational trophoblastic neoplasia (GTN). Normal prostatic tissue and Breast cancer tissue were used as positive controls. Results We studied 28 patients with simple molar pregnancy (SMP) and 30 with GTN. Cytotrophobalst had significantly higher expression of p53 and c-erbB-2 and syncytiotrophoblast had greater expression of p53 in GTN group as compared to SMP group. The cut off values for percentage of p53 positive immunostained cytotrophoblast and syncytiotrophoblast were 5.5% and 2.5%. In c-erbB-2 positive membranous stained cytotrophoblast the cut off was 12.5%. Conclusions Our data suggests that over expression of p53 and c-erbB-2 is associated with malignant progression of molar pregnancy. We encountered that high expression of p53 and c-erbB-2 in trophoblastic cells could predict gestational trophoblastic neoplasia during the early stages. PMID:27703642

  14. Integrated Amplification Microarrays for Infectious Disease Diagnostics

    PubMed Central

    Chandler, Darrell P.; Bryant, Lexi; Griesemer, Sara B.; Gu, Rui; Knickerbocker, Christopher; Kukhtin, Alexander; Parker, Jennifer; Zimmerman, Cynthia; George, Kirsten St.; Cooney, Christopher G.

    2012-01-01

    This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.

  15. Microarray data analysis and mining approaches.

    PubMed

    Cordero, Francesca; Botta, Marco; Calogero, Raffaele A

    2007-12-01

    Microarray based transcription profiling is now a consolidated methodology and has widespread use in areas such as pharmacogenomics, diagnostics and drug target identification. Large-scale microarray studies are also becoming crucial to a new way of conceiving experimental biology. A main issue in microarray transcription profiling is data analysis and mining. When microarrays became a methodology of general use, considerable effort was made to produce algorithms and methods for the identification of differentially expressed genes. More recently, the focus has switched to algorithms and database development for microarray data mining. Furthermore, the evolution of microarray technology is allowing researchers to grasp the regulative nature of transcription, integrating basic expression analysis with mRNA characteristics, i.e. exon-based arrays, and with DNA characteristics, i.e. comparative genomic hybridization, single nucleotide polymorphism, tiling and promoter structure. In this article, we will review approaches used to detect differentially expressed genes and to link differential expression to specific biological functions.

  16. Imaging the morphological change of tissue structure during the early phase of esophageal tumor progression using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Jian; Kang, Deyong; Xu, Meifang; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Jianxin

    2012-12-01

    Esophageal cancer is a common malignancy with a very poor prognosis. Successful strategies for primary prevention and early detection are critically needed to control this disease. Multiphoton microscopy (MPM) is becoming a novel optical tool of choice for imaging tissue architecture and cellular morphology by two-photon excited fluorescence. In this study, we used MPM to image microstructure of human normal esophagus, carcinoma in situ (CIS), and early invasive carcinoma in order to establish the morphological features to differentiate these tissues. The diagnostic features such as the appearance of cancerous cells, the significant loss of stroma, the absence of the basement membrane were extracted to distinguish between normal and cancerous esophagus tissue. These results correlated well with the paired histological findings. With the advancement of clinically miniaturized MPM and the multi-photon probe, combining MPM with standard endoscopy will therefore allow us to make a real-time in vivo diagnosis of early esophageal cancer at the cellular level.

  17. Automated Microarray Image Analysis Toolbox for MATLAB

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Willse, Alan R.; Protic, Miroslava; Chandler, Darrell P.

    2005-09-01

    The Automated Microarray Image Analysis (AMIA) Toolbox for MATLAB is a flexible, open-source microarray image analysis tool that allows the user to customize analysis of sets of microarray images. This tool provides several methods of identifying and quantify spot statistics, as well as extensive diagnostic statistics and images to identify poor data quality or processing. The open nature of this software allows researchers to understand the algorithms used to provide intensity estimates and to modify them easily if desired.

  18. Surface free energy and microarray deposition technology.

    PubMed

    McHale, Glen

    2007-03-01

    Microarray techniques use a combinatorial approach to assess complex biochemical interactions. The fundamental goal is simultaneous, large-scale experimentation analogous to the automation achieved in the semiconductor industry. However, microarray deposition inherently involves liquids contacting solid substrates. Liquid droplet shapes are determined by surface and interfacial tension forces, and flows during drying. This article looks at how surface free energy and wetting considerations may influence the accuracy and reliability of spotted microarray experiments.

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

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-06

    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.

  20. Detect Key Gene Information in Classification of Microarray Data

    NASA Astrophysics Data System (ADS)

    Liu, Yihui

    2008-12-01

    We detect key information of high-dimensional microarray profiles based on wavelet analysis and genetic algorithm. Firstly, wavelet transform is employed to extract approximation coefficients at 2nd level, which remove noise and reduce dimensionality. Genetic algorithm (GA) is performed to select the optimized features. Experiments are performed on four datasets, and experimental results prove that approximation coefficients are efficient way to characterize the microarray data. Furthermore, in order to detect the key genes in the classification of cancer tissue, we reconstruct the approximation part of gene profiles based on orthogonal approximation coefficients. The significant genes are selected based on reconstructed approximation information using genetic algorithm. Experiments prove that good performance of classification is achieved based on the selected key genes.

  1. Discovering cancer biomarkers from clinical samples by protein microarrays.

    PubMed

    Hu, Bin; Niu, Xin; Cheng, Li; Yang, Li-Na; Li, Qing; Wang, Yang; Tao, Sheng-Ce; Zhou, Shu-Min

    2015-02-01

    Cancer biomarkers are of potential use in early cancer diagnosis, anticancer therapy development, and monitoring the responses to treatments. Protein-based cancer biomarkers are major forms in use, as they are much easier to be monitored in body fluids or tissues. For cancer biomarker discovery, high-throughput techniques such as protein microarrays hold great promises, because they are capable of global unbiased monitoring but with a miniaturized format. In doing so, novel and cancer type specific biomarkers can be systematically discovered at an affordable cost. In this review, we give a relatively complete picture on protein microarrays applied to clinical samples for cancer biomarker discovery, and conclude this review with the future perspectives. PMID:25523829

  2. Reverse phase protein microarrays advance to use in clinical trials

    PubMed Central

    Mueller, Claudius; Liotta, Lance A.; Espina, Virginia

    2010-01-01

    Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis. PMID:20974554

  3. 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...

  4. Living Cell Microarrays: An Overview of Concepts.

    PubMed

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

  5. Living Cell Microarrays: An Overview of Concepts

    PubMed Central

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

  6. Living Cell Microarrays: An Overview of Concepts

    PubMed Central

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.

  7. Highly parallel microbial diagnostics using oligonucleotide microarrays.

    PubMed

    Loy, Alexander; Bodrossy, Levente

    2006-01-01

    Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simultaneous identification of many different microorganisms and viruses in a single assay. In the past few years, researchers have been confronted with a dramatic increase in the number of studies reporting development and/or improvement of oligonucleotide microarrays for microbial diagnostics, but use of the technology in routine diagnostics is still constrained by a variety of factors. Careful development of microarray essentials (such as oligonucleotide probes, protocols for target preparation and hybridization, etc.) combined with extensive performance testing are thus mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely applicable tools.

  8. A methodical microarray design enables surveying of expression of a broader range of genes in Ciona intestinalis.

    PubMed

    Matsumae, Hiromi; Hamada, Mayuko; Fujie, Manabu; Niimura, Yoshihito; Tanaka, Hiroshi; Kawashima, Takeshi

    2013-04-25

    We provide a new oligo-microarray for Ciona intestinalis, based on the NimbleGen 12-plex×135k format. The array represents 106,285 probes, which is more than double the probe number of the currently available 44k microarray. These probes cover 99.2% of the transcripts in the KyotoHoya (KH) models, published in 2008, and they contain 81.1% of the entries in the UniGene database that are not included in the KH models. In this paper, we show that gene expression levels measured by this new 135k microarray are highly correlated with those obtained by the existing 44k microarray for genes common to both arrays. We also investigated gene expression using samples obtained from the ovary and the neural complex of adult C. intestinalis, showing that the expression of tissue-specific genes is consistent with previous reports. Approximately half of the highly expressed genes identified in the 135k microarray are not included in the previous microarray. The high coverage of gene models by this microarray made it possible to identify splicing variants for a given transcript. The 135k microarray is useful in investigating the functions of genes that are not yet well characterized. Detailed information about this 135k microarray is accessible at no charge from supplemental materials, NCBI Gene Expression Omnibus (GEO), and http://marinegenomics.oist.jp.

  9. 2008 Microarray Research Group (MARG Survey): Sensing the State of Microarray Technology

    EPA Science Inventory

    Over the past several years, the field of microarrays has grown and evolved drastically. In its continued efforts to track this evolution and transformation, the ABRF-MARG has once again conducted a survey of international microarray facilities and individual microarray users. Th...

  10. THE ABRF-MARG MICROARRAY SURVEY 2004: TAKING THE PULSE OF THE MICROARRAY FIELD

    EPA Science Inventory

    Over the past several years, the field of microarrays has grown and evolved drastically. In its continued efforts to track this evolution, the ABRF-MARG has once again conducted a survey of international microarray facilities and individual microarray users. The goal of the surve...

  11. Microarray analysis in caudal medulla of cattle orally challenged with bovine spongiform encephalopathy.

    PubMed

    Almeida, L M; Basu, U; Williams, J L; Moore, S S; Guan, L L

    2011-10-25

    Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, amphetamine-responsive transcript, and synaptosomal-associated protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.

  12. Recent progress in defining mechanisms and potential targets for prevention of normal tissue injury after radiation therapy

    SciTech Connect

    Anscher, Mitchell S. . E-mail: anscher@radonc.duke.edu; Chen, Liguang; Rabbani, Zahid; Kang Song; Larrier, Nicole; Huang Hong; Samulski, Thaddeus V.; Dewhirst, Mark W.; Brizel, David M.; Folz, Rodney J.; Vujaskovic, Zeljko

    2005-05-01

    The ability to optimize treatments for cancer on the basis of relative risks for normal tissue injury has important implications in oncology, because higher doses of radiation might, in some diseases, improve both local control and survival. To achieve this goal, a thorough understanding of the molecular mechanisms responsible for radiation-induced toxicity will be essential. Recent research has demonstrated that ionizing radiation triggers a series of genetic and molecular events, which might lead to chronic persistent alterations in the microenvironment and an aberrant wound-healing response. Disrupted epithelial-stromal cell communication might also be important. With the application of a better understanding of fundamental biology to clinical practice, new approaches to treating and preventing normal tissue injury can focus on correcting these disturbed molecular processes.

  13. Proton irradiation impacts age-driven modulations of cancer progression influenced by immune system transcriptome modifications from splenic tissue

    PubMed Central

    Wage, Justin; Ma, Lili; Peluso, Michael; Lamont, Clare; Evens, Andrew M.; Hahnfeldt, Philip; Hlatky, Lynn; Beheshti, Afshin

    2015-01-01

    Age plays a crucial role in the interplay between tumor and host, with additional impact due to irradiation. Proton irradiation of tumors induces biological modulations including inhibition of angiogenic and immune factors critical to ‘hallmark’ processes impacting tumor development. Proton irradiation has also provided promising results for proton therapy in cancer due to targeting advantages. Additionally, protons may contribute to the carcinogenesis risk from space travel (due to the high proportion of high-energy protons in space radiation). Through a systems biology approach, we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice was altered with age, with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68-day) versus old (736-day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5 Gy (1-GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice, as compared with older subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2, MCM7, CD74 and RUVBL2 indicated these were the key genes involved in the regulatory changes in the host environment response (i.e. the spleen). Collectively, these results suggest that a significant biological component of proton irradiation is modulated by host age through promotion of carcinogenesis in adolescence and resistance to immunosuppression, carcinogenesis and genetic perturbation associated with advancing age. PMID:26253138

  14. Proton irradiation impacts age-driven modulations of cancer progression influenced by immune system transcriptome modifications from splenic tissue.

    PubMed

    Wage, Justin; Ma, Lili; Peluso, Michael; Lamont, Clare; Evens, Andrew M; Hahnfeldt, Philip; Hlatky, Lynn; Beheshti, Afshin

    2015-09-01

    Age plays a crucial role in the interplay between tumor and host, with additional impact due to irradiation. Proton irradiation of tumors induces biological modulations including inhibition of angiogenic and immune factors critical to 'hallmark' processes impacting tumor development. Proton irradiation has also provided promising results for proton therapy in cancer due to targeting advantages. Additionally, protons may contribute to the carcinogenesis risk from space travel (due to the high proportion of high-energy protons in space radiation). Through a systems biology approach, we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice was altered with age, with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68-day) versus old (736-day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5 Gy (1-GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice, as compared with older subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2, MCM7, CD74 and RUVBL2 indicated these were the key genes involved in the regulatory changes in the host environment response (i.e. the spleen). Collectively, these results suggest that a significant biological component of proton irradiation is modulated by host age through promotion of carcinogenesis in adolescence and resistance to immunosuppression, carcinogenesis and genetic perturbation associated with advancing age.

  15. Noninvasive near-infrared fluorescent protein-based imaging of tumor progression and metastases in deep organs and intraosseous tissues

    NASA Astrophysics Data System (ADS)

    Jiguet-Jiglaire, Carine; Cayol, Mylène; Mathieu, Sylvie; Jeanneau, Charlotte; Bouvier-Labit, Corinne; Ouafik, L.'houcine; El-Battari, Assou

    2014-01-01

    Whole-body imaging of experimental tumor growth is more feasible within the near-infrared (NIR) optical window because of the highest transparency of mammalian tissues within this wavelength spectrum, mainly due to improved tissue penetration and lower autofluorescence. We took advantage from the recently cloned infrared fluorescent protein (iRFP) together with a human immunodeficiency virus (HIV)-based lentiviral vector to produce virally transduced tumor cells that permanently express this protein. We then noninvasively explored metastatic spread as well as primary tumor growth in deep organs and behind bone barriers. Intrabone tumor growth was investigated through intracranial and intratibial injections of glioblastoma and osteosarcoma cells, respectively, and metastasis was assessed by tail vein injection of melanoma cells. We found that the emitted fluorescence is captured as sharp images regardless of the organ or tissue considered. Furthermore, by overlaying fluorescence spots with the white light, it was possible to afford whole-body images yet never observed before. This approach allowed us to continuously monitor the growth and dissemination of tumor cells with a small number of animals, minimal animal handling, and without the need for any additive. This iRFP-based system provides high-resolution readouts of tumorigenesis that should greatly facilitate preclinical trials with anticancer therapeutic molecules.

  16. Progress in theoretical, experimental, and computational investigations in turbid tissue phantoms and human teeth using laser infrared photothermal radiometry

    NASA Astrophysics Data System (ADS)

    Mandelis, Andreas

    2002-03-01

    This paper reviews and describes the state-of-the-art in the development of frequency-domain infrared photothermal radiometry (FD-PTR) for biomedical and dental applications. The emphasis is placed on the measurement of the optical and thermal properties of tissue-like materials using FD-PTR. A rigorous three-dimensional thermal-wave formulation with three-dimensional diffuse and coherent photon-density-wave sources is presented, and is applied to data from model tissue phantoms and dental enamel samples. The combined theoretical, experimental and computational methodology shows good promise with regard to its analytical ability to measure optical properties of turbid media uniquely, as compared to PPTR, which exhibits uniqueness problems. From data sets obtained with calibrated test phantoms, the reduced optical scattering and absorption coefficients were found to be within 20% and 10%, respectively, from the independently derived values using Mie scattering theory and spectrophotometric measurements. Furthermore, the state-of-the-art and recent developments in applications of laser infrared FD-PTR to dental caries research is described, with examples and histological studies from carious dental tissue. The correlation of PTR signals with modulated dental luminescence is discussed as a very promising potential quantitative methodology for the clinical diagnosis of sub-surface incipient dental caries. The application of the turbid-medium thermal-wave model to the measurement of the optical absorption and scattering coefficients of enamel is also presented.

  17. Polyanionic collagen membranes for guided tissue regeneration: Effect of progressive glutaraldehyde cross-linking on biocompatibility and degradation.

    PubMed

    Veríssimo, D M; Leitão, R F C; Ribeiro, R A; Figueiró, S D; Sombra, A S B; Góes, J C; Brito, G A C

    2010-10-01

    The ultimate goal of periodontal therapy is to control periodontal tissue inflammation and to produce predictable regeneration of that part of the periodontium which has been lost as a result of periodontal disease. In guided tissue regeneration membranes function as mechanical barriers, excluding the epithelium and gingival corium from the root surface and allowing regeneration by periodontal ligament cells. This report aims to study the effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen (PAC) membranes by conducting a histological evaluation of the tissue response (biocompatibility) and by assessing the biodegradation of subcutaneous membrane implants in rats. We studied six different samples: a PAC, a PAC mineralized by alternate soaking processes for either 25 or 75 cycles (PAC 25 and PAC 75, respectively) and these films cross-linked by GA. Inflammatory infiltrate, cytokine dosage, fibrosis capsule thickness, metalloproteinase immunohistochemistry and membrane biodegradation after 1, 7, 15 and 30 days were measured. The inflammatory response was found to be more intense in membranes without cross-linking, while the fibrosis capsules became thicker in cross-linked membranes after 30 days. The membranes without cross-linking suffered intense biodegradation, while the membranes with cross-linking remained intact after 30 days. The cross-linking with GA reduced the inflammatory response and prevented degradation of the membranes over the entire course of the observation period. These membranes are thus an attractive option when the production of new bone depends on the prolonged presence of a mechanical barrier.

  18. Noninvasive near-infrared fluorescent protein-based imaging of tumor progression and metastases in deep organs and intraosseous tissues.

    PubMed

    Jiguet-Jiglaire, Carine; Cayol, Mylène; Mathieu, Sylvie; Jeanneau, Charlotte; Bouvier-Labit, Corinne; Ouafik, L'houcine; El-Battari, Assou

    2014-01-01

    Whole-body imaging of experimental tumor growth is more feasible within the near-infrared (NIR) optical window because of the highest transparency of mammalian tissues within this wavelength spectrum, mainly due to improved tissue penetration and lower autofluorescence. We took advantage from the recently cloned infrared fluorescent protein (iRFP) together with a human immunodeficiency virus (HIV)-based lentiviral vector to produce virally transduced tumor cells that permanently express this protein. We then noninvasively explored metastatic spread as well as primary tumor growth in deep organs and behind bone barriers. Intrabone tumor growth was investigated through intracranial and intratibial injections of glioblastoma and osteosarcoma cells, respectively, and metastasis was assessed by tail vein injection of melanoma cells. We found that the emitted fluorescence is captured as sharp images regardless of the organ or tissue considered. Furthermore, by overlaying fluorescence spots with the white light, it was possible to afford whole-body images yet never observed before. This approach allowed us to continuously monitor the growth and dissemination of tumor cells with a small number of animals, minimal animal handling, and without the need for any additive. This iRFP-based system provides high-resolution readouts of tumorigenesis that should greatly facilitate preclinical trials with anticancer therapeutic molecules. PMID:24474505

  19. Ontology-Based Analysis of Microarray Data.

    PubMed

    Giuseppe, Agapito; Milano, Marianna

    2016-01-01

    The importance of semantic-based methods and algorithms for the analysis and management of biological data is growing for two main reasons. From a biological side, knowledge contained in ontologies is more and more accurate and complete, from a computational side, recent algorithms are using in a valuable way such knowledge. Here we focus on semantic-based management and analysis of protein interaction networks referring to all the approaches of analysis of protein-protein interaction data that uses knowledge encoded into biological ontologies. Semantic approaches for studying high-throughput data have been largely used in the past to mine genomic and expression data. Recently, the emergence of network approaches for investigating molecular machineries has stimulated in a parallel way the introduction of semantic-based techniques for analysis and management of network data. The application of these computational approaches to the study of microarray data can broad the application scenario of them and simultaneously can help the understanding of disease development and progress.

  20. Studying bovine early embryo transcriptome by microarray.

    PubMed

    Dufort, Isabelle; Robert, Claude; Sirard, Marc-André

    2015-01-01

    Microarrays represent a significant advantage when studying gene expression in early embryo because they allow for a speedy study of a large number of genes even if the sample of interest contains small quantities of genetic material. Here we describe the protocols developed by the EmbryoGENE Network to study the bovine transcriptome in early embryo using a microarray experimental design.

  1. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  2. Application of microarray technology in pulmonary diseases

    PubMed Central

    Tzouvelekis, Argyris; Patlakas, George; Bouros, Demosthenes

    2004-01-01

    Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives. PMID:15585067

  3. Sensing immune responses with customized peptide microarrays.

    PubMed

    Schirwitz, Christopher; Loeffler, Felix F; Felgenhauer, Thomas; Stadler, Volker; Breitling, Frank; Bischoff, F Ralf

    2012-12-01

    The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

  4. Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis.

    PubMed

    Schinköthe, Jan; Köhler, Heike; Liebler-Tenorio, Elisabeth M

    2016-08-01

    Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4(+), CD8(+), γδ, B lymphocytes and plasma, CD25(+), CD68(+), MHC-II(+), Ki67(+) and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4(+) T cells, but only few epitheloid macrophages were observed in severely sick goats at 2-3mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research. PMID:27477506

  5. Extracellular Matrix, Nuclear and Chromatin Structure and GeneExpression in Normal Tissues and Malignant Tumors: A Work inProgress

    SciTech Connect

    Spencer, Virginia A.; Xu, Ren; Bissell, Mina J.

    2006-08-01

    Almost three decades ago, we presented a model where theextracellular matrix (ECM) was postulated to influence gene expressionand tissue-specificity through the action of ECM receptors and thecytoskeleton. This hypothesis implied that ECM molecules could signal tothe nucleus and that the unit of function in higher organisms was not thecell alone, but the cell plus its microenvironment. We now know that ECMinvokes changes in tissue and organ architecture and that tissue, cell,nuclear, and chromatin structure are changed profoundly as a result ofand during malignant progression. Whereas some evidence has beengenerated for a link between ECM-induced alterations in tissuearchitecture and changes in both nuclear and chromatin organization, themanner by which these changes actively induce or repress gene expressionin normal and malignant cells is a topic in need of further attention.Here, we will discuss some key findings that may provide insights intomechanisms through which ECM could influence gene transcription and howtumor cells acquire the ability to overcome these levels ofcontrol.

  6. Microarray Applications in Microbial Ecology Research.

    SciTech Connect

    Gentry, T.; Schadt, C.; Zhou, J.

    2006-04-06

    Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. This review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.

  7. Real-time DNA microarray analysis

    PubMed Central

    Hassibi, Arjang; Vikalo, Haris; Riechmann, José Luis; Hassibi, Babak

    2009-01-01

    We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays. PMID:19723688

  8. Real-time DNA microarray analysis.

    PubMed

    Hassibi, Arjang; Vikalo, Haris; Riechmann, José Luis; Hassibi, Babak

    2009-11-01

    We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays. PMID:19723688

  9. Fusion transcript discovery in formalin-fixed paraffin-embedded human breast cancer tissues reveals a link to tumor progression.

    PubMed

    Ma, Yan; Ambannavar, Ranjana; Stephans, James; Jeong, Jennie; Dei Rossi, Andrew; Liu, Mei-Lan; Friedman, Adam J; Londry, Jason J; Abramson, Richard; Beasley, Ellen M; Baker, Joffre; Levy, Samuel; Qu, Kunbin

    2014-01-01

    The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.

  10. Integrated imaging instrument for self-calibrated fluorescence protein microarrays

    NASA Astrophysics Data System (ADS)

    Reddington, A. P.; Monroe, M. R.; Ünlü, M. S.

    2013-10-01

    Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

  11. DNA Microarrays for Identifying Fishes

    PubMed Central

    Nölte, M.; Weber, H.; Silkenbeumer, N.; Hjörleifsdottir, S.; Hreggvidsson, G. O.; Marteinsson, V.; Kappel, K.; Planes, S.; Tinti, F.; Magoulas, A.; Garcia Vazquez, E.; Turan, C.; Hervet, C.; Campo Falgueras, D.; Antoniou, A.; Landi, M.; Blohm, D.

    2008-01-01

    In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products. PMID:18270778

  12. 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.

  13. Delivering Nucleic-Acid Based Nanomedicines on Biomaterial Scaffolds for Orthopedic Tissue Repair: Challenges, Progress and Future Perspectives.

    PubMed

    Raftery, Rosanne M; Walsh, David P; Castaño, Irene Mencía; Heise, Andreas; Duffy, Garry P; Cryan, Sally-Ann; O'Brien, Fergal J

    2016-07-01

    As well as acting to fill defects and allow for cell infiltration and proliferation in regenerative medicine, biomaterial scaffolds can also act as carriers for therapeutics, further enhancing their efficacy. Drug and protein delivery on scaffolds have shown potential, however, supraphysiological quantities of therapeutic are often released at the defect site, causing off-target side effects and cytotoxicity. Gene therapy involves the introduction of foreign genes into a cell in order to exert an effect; either replacing a missing gene or modulating expression of a protein. State of the art gene therapy also encompasses manipulation of the transcriptome by harnessing RNA interference (RNAi) therapy. The delivery of nucleic acid nanomedicines on biomaterial scaffolds - gene-activated scaffolds -has shown potential for use in a variety of tissue engineering applications, but as of yet, have not reached clinical use. The current state of the art in terms of biomaterial scaffolds and delivery vector materials for gene therapy is reviewed, and the limitations of current procedures discussed. Future directions in the clinical translation of gene-activated scaffolds are also considered, with a particular focus on bone and cartilage tissue regeneration.

  14. Delivering Nucleic-Acid Based Nanomedicines on Biomaterial Scaffolds for Orthopedic Tissue Repair: Challenges, Progress and Future Perspectives.

    PubMed

    Raftery, Rosanne M; Walsh, David P; Castaño, Irene Mencía; Heise, Andreas; Duffy, Garry P; Cryan, Sally-Ann; O'Brien, Fergal J

    2016-07-01

    As well as acting to fill defects and allow for cell infiltration and proliferation in regenerative medicine, biomaterial scaffolds can also act as carriers for therapeutics, further enhancing their efficacy. Drug and protein delivery on scaffolds have shown potential, however, supraphysiological quantities of therapeutic are often released at the defect site, causing off-target side effects and cytotoxicity. Gene therapy involves the introduction of foreign genes into a cell in order to exert an effect; either replacing a missing gene or modulating expression of a protein. State of the art gene therapy also encompasses manipulation of the transcriptome by harnessing RNA interference (RNAi) therapy. The delivery of nucleic acid nanomedicines on biomaterial scaffolds - gene-activated scaffolds -has shown potential for use in a variety of tissue engineering applications, but as of yet, have not reached clinical use. The current state of the art in terms of biomaterial scaffolds and delivery vector materials for gene therapy is reviewed, and the limitations of current procedures discussed. Future directions in the clinical translation of gene-activated scaffolds are also considered, with a particular focus on bone and cartilage tissue regeneration. PMID:26840618

  15. Chromatin immunoprecipitation and microarray-based analysis of protein location

    PubMed Central

    Lee, Tong Ihn; Johnstone, Sarah E; Young, Richard A

    2010-01-01

    Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types. PMID:17406303

  16. Fecal source tracking in water using a mitochondrial DNA microarray.

    PubMed

    Vuong, Nguyet-Minh; Villemur, Richard; Payment, Pierre; Brousseau, Roland; Topp, Edward; Masson, Luke

    2013-01-01

    A mitochondrial-based microarray (mitoArray) was developed for rapid identification of the presence of 28 animals and one family (cervidae) potentially implicated in fecal pollution in mixed activity watersheds. Oligonucleotide probes for genus or subfamily-level identification were targeted within the 12S rRNA - Val tRNA - 16S rRNA region in the mitochondrial genome. This region, called MI-50, was selected based on three criteria: 1) the ability to be amplified by universal primers 2) these universal primer sequences are present in most commercial and domestic animals of interest in source tracking, and 3) that sufficient sequence variation exists within this region to meet the minimal requirements for microarray probe discrimination. To quantify the overall level of mitochondrial DNA (mtDNA) in samples, a quantitative-PCR (Q-PCR) universal primer pair was also developed. Probe validation was performed using DNA extracted from animal tissues and, for many cases, animal-specific fecal samples. To reduce the amplification of potentially interfering fish mtDNA sequences during the MI-50 enrichment step, a clamping PCR method was designed using a fish-specific peptide nucleic acid. DNA extracted from 19 water samples were subjected to both array and independent PCR analyses. Our results confirm that the mitochondrial microarray approach method could accurately detect the dominant animals present in water samples emphasizing the potential for this methodology in the parallel scanning of a large variety of animals normally monitored in fecal source tracking.

  17. Lung cancer in uranium miners: A tissue resource and pilot study. Progress report, September 25, 1992--May 31, 1993

    SciTech Connect

    Samet, J.M.

    1993-05-01

    This project involves two related activities directed toward understanding respiratory carcinogenesis in radon-exposed former uranium miners. The first activity involves a continuation of the tissue resource of lung cancer cases from former underground uranium miners and comparison cases from non-miners. The second activity is a pilot study for a proposed longitudinal study of respiratory carcinogenesis in former uranium miners. The objectives are to facilitate the investigation of molecular changes in radon exposed lung cancer cases and to develop methods for prospectively studying clinical, cytologic, cytogenetic, and molecular changes in the multi-event process of respiratory carcinogenesis, and to assess the feasibility of recruiting former uranium miners into a longitudinal study that collects multiple biologic specimens.

  18. Intra-parenchymal ferrous iron infusion causes neuronal atrophy, cell death and progressive tissue loss: implications for intracerebral hemorrhage.

    PubMed

    Caliaperumal, Jayalakshmi; Ma, Yonglie; Colbourne, Frederick

    2012-10-01

    Intracerebral hemorrhage (ICH) is a devastating stroke causing considerable tissue destruction from mechanical trauma and secondary degeneration. Free iron, released over days from degrading erythrocytes, causes free radicals that likely contribute to delayed injury. Indeed, an intracerebral injection of iron rapidly kills cells and causes cerebral edema. We expanded upon these observations by: determining a dose-response relationship of iron infusion, examining the structural appearance of surviving striatal neurons, and evaluating injury over months. First, we measured 24-h edema in rats given 3.8, 19.0 or 38.0 μg infusions of FeCl₂ (i.e., 30 μL of a 1, 5 or 10 mmol/L solution). Second, rats were given these infusions (vs. saline controls) followed by behavioral assessment and histology at 7 days. Third, dendritic structure was measured in Golgi-Cox stained neurons at 7 days after a 0.95-μg dose (30 μL of a 0.25 mmol/L solution). Last, rats survived 7 or 60 days post-injection (19.0 μg) for histological assessment. Larger doses of iron caused greater injury, but this was generally not reflected in behavior that indicated similar deficits among the 3.8-38.0 μg groups. Similarly, edema occurred but was not linearly related to dose. Even after a low iron dose the surviving neurons in the peri-injury zone were considerably atrophied (vs. contralateral side and controls). Finally, continuing tissue loss occurred over weeks with prominent neuronal death and iron-positive cells (e.g., macrophages) at 60 days. Iron alone may account for the chronic degeneration found after ICH in rodent models.

  19. Progression from high insulin resistance to type 2 diabetes does not entail additional visceral adipose tissue inflammation.

    PubMed

    Barbarroja, Nuria; Lopez-Pedrera, Chary; Garrido-Sanchez, Lourdes; Mayas, Maria Dolores; Oliva-Olivera, Wilfredo; Bernal-Lopez, Maria Rosa; El Bekay, Rajaa; Tinahones, Francisco Jose

    2012-01-01

    Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.

  20. Progression from High Insulin Resistance to Type 2 Diabetes Does Not Entail Additional Visceral Adipose Tissue Inflammation

    PubMed Central

    Barbarroja, Nuria; Lopez-Pedrera, Chary; Garrido-Sanchez, Lourdes; Mayas, Maria Dolores; Oliva-Olivera, Wilfredo; Bernal-Lopez, Maria Rosa; El Bekay, Rajaa; Tinahones, Francisco Jose

    2012-01-01

    Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity. PMID:23110196

  1. Microarray-integrated optoelectrofluidic immunoassay system.

    PubMed

    Han, Dongsik; Park, Je-Kyun

    2016-05-01

    A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

  2. Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry

    PubMed Central

    McKee, Christopher J.; Hines, Harry B.; Ulrich, Robert G.

    2013-01-01

    Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein-protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein-peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a pre-defined raster of MALDI-TOF MS spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium-tin oxide. Further, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI-TOF instruments and has general utility for the label-free analysis of microarray assays. PMID:23906642

  3. Systematic review of accuracy of prenatal diagnosis for abnormal chromosome diseases by microarray technology.

    PubMed

    Xu, H B; Yang, H; Liu, G; Chen, H

    2014-10-31

    The accuracy of prenatal diagnosis for abnormal chromosome diseases by chromosome microarray technology and karyotyping were compared. A literature search was carried out in the MEDLINE database with the keywords "chromosome" and "karyotype" and "genetic testing" and "prenatal diagnosis" and "oligonucleotide array sequence". The studies obtained were filtered by using the QUADAS tool, and studies conforming to the quality standard were fully analyzed. There was one paper conforming to the QUADAS standards including 4406 gravidas with adaptability syndromes of prenatal diagnosis including elderly parturient women, abnormal structure by type-B ultrasound, and other abnormalities. Microarray technology yielded successful diagnoses in 4340 cases (98.8%), and there was no need for tissue culture in 87.9% of the samples. All aneuploids and non-parallel translocations in 4282 cases of non-chimera identified by karyotyping could be detected using microarray analysis technology, whereas parallel translocations and fetal triploids could not be detected by microarray analysis technology. In the samples with normal karyotyping results, type-B ultrasound showed that 6% of chromosomal deficiencies or chromosome duplications could be detected by microarray technology, and the same abnormal chromosomes were detected in 1.7% of elderly parturient women and samples with positive serology screening results. In the prenatal diagnosis test, compared with karyotyping, microarray technology could identify the extra cell genetic information with clinical significance, aneuploids, and non-parallel translocations; however, its disadvantage is that it could not identify parallel translocations and triploids.

  4. 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.

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

    PubMed Central

    Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

    2016-01-01

    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. PMID:27657141

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

    PubMed

    Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

    2016-01-01

    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. PMID:27657141

  7. Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.

    PubMed

    Dittami, Simon M; Hostyeva, Vladyslava; Egge, Elianne Sirnæs; Kegel, Jessica U; Eikrem, Wenche; Edvardsen, Bente

    2013-10-01

    Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring.

  8. Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.

    PubMed

    Dittami, Simon M; Hostyeva, Vladyslava; Egge, Elianne Sirnæs; Kegel, Jessica U; Eikrem, Wenche; Edvardsen, Bente

    2013-10-01

    Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring. PMID:23325054

  9. DNA Microarrays in Herbal Drug Research

    PubMed Central

    Chavan, Preeti; Joshi, Kalpana; Patwardhan, Bhushan

    2006-01-01

    Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts. PMID:17173108

  10. Development and function of membrane systems in plant tissue. Annual technical progress report, 15 September 1981-15 August 1982

    SciTech Connect

    Hanson, J B

    1982-01-01

    Over the past 11 months we have continued investigation of ion transport mechanisms in corn roots and mitochondria. In mitochondria we find that only citrate and isocitrate are transported by the H/sup +//citrate symporter. However, the in vivo function of this carrier remains in doubt because citrate does not appear to be an effective substrate for corn mitochondria. Studies with roots have been directed to why various types of injury or shock all result in temporary blockage of the H/sup +/-efflux pump in the plasmamembrane. It appears this may be due to an injury-mediated Ca/sup 2 +/ influx into the tissue, which by raising free Ca/sup 2 +/ in the cytosal activates calmodulin (CaM). In turn, the Ca.CaM complex appears to activate protein kinase, phosphorylating membrane proteins. It is possible that one of these phosphorylated proteins is responsible for inactivation of the H/sup +/-ATPase. Future work is planned around the consequences of Ca/sup 2 +/ influx into the root cell subsequent to injury, investigating the recovery of the H/sup +/-ATPase and the initiation of the biosyntheses which lead to augmented ion transport.

  11. Tissue Inhibitor of Metalloproteinase-1 Is Confined to Tumor-Associated Myofibroblasts and Is Increased With Progression in Gastric Adenocarcinoma.

    PubMed

    Alpízar-Alpízar, Warner; Laerum, Ole Didrik; Christensen, Ib J; Ovrebo, Kjell; Skarstein, Arne; Høyer-Hansen, Gunilla; Ploug, Michael; Illemann, Martin

    2016-08-01

    The tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the extracellular matrix-degrading activity of several matrix metalloproteinases, thereby regulating cancer cell invasion and metastasis. Studies describing the expression pattern and cellular localization of TIMP-1 in gastric cancer are, however, highly discordant. We addressed these inconsistencies by performing immunohistochemistry and in situ hybridization analyses in a set of 49 gastric cancer lesions to reexamine the TIMP-1 localization. In addition, we correlated these findings to clinicopathological parameters. We show that strong expression of TIMP-1 protein and mRNA was observed in a subpopulation of stromal fibroblast-like cells at the periphery of the cancer lesions. In a few cases, a small fraction of cancer cells showed weak expression of TIMP-1 protein and mRNA. The stromal TIMP-1-expressing cells were mainly tumor-associated myofibroblasts. In the normal-appearing mucosa, scattered TIMP-1 protein was only found in chromogranin A positive cells. TIMP-1-positive myofibroblasts at the invasive front of the tumors were more frequently seen in intestinal than in diffuse histological subtype cases (p=0.009). A significant trend to a higher number of cases showing TIMP-1 staining in myofibroblasts with increasing tumor, node, metastasis (TNM) stage was also revealed (p=0.041). In conclusion, tumor-associated myofibroblasts are the main source of increased TIMP-1 expression in gastric cancer. PMID:27370797

  12. Melanoma Prognostic Model Using Tissue Microarrays and Genetic Algorithms

    PubMed Central

    Gould Rothberg, Bonnie E.; Berger, Aaron J.; Molinaro, Annette M.; Subtil, Antonio; Krauthammer, Michael O.; Camp, Robert L.; Bradley, William R.; Ariyan, Stephan; Kluger, Harriet M.; Rimm, David L.

    2009-01-01

    Purpose As a result of the questionable risk-to-benefit ratio of adjuvant therapies, stage II melanoma is currently managed by observation because available clinicopathologic parameters cannot identify the 20% to 60% of such patients likely to develop metastatic disease. Here, we propose a multimarker molecular prognostic assay that can help triage patients at increased risk of recurrence. Methods Protein expression for 38 candidates relevant to melanoma oncogenesis was evaluated using the automated quantitative analysis (AQUA) method for immunofluorescence-based immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a cohort of 192 primary melanomas collected during 1959 to 1994. The prognostic assay was built using a genetic algorithm and validated on an independent cohort of 246 serial primary melanomas collected from 1997 to 2004. Results Multiple iterations of the genetic algorithm yielded a consistent five-marker solution. A favorable prognosis was predicted by ATF2 ln(non-nuclear/nuclear AQUA score ratio) of more than –0.052, p21WAF1 nuclear compartment AQUA score of more than 12.98, p16INK4A ln(non-nuclear/nuclear AQUA score ratio) of ≤ −0.083, β-catenin total AQUA score of more than 38.68, and fibronectin total AQUA score of ≤ 57.93. Primary tumors that met at least four of these five conditions were considered a low-risk group, and those that met three or fewer conditions formed a high-risk group (log-rank P < .0001). Multivariable proportional hazards analysis adjusting for clinicopathologic parameters shows that the high-risk group has significantly reduced survival on both the discovery (hazard ratio = 2.84; 95% CI, 1.46 to 5.49; P = .002) and validation (hazard ratio = 2.72; 95% CI, 1.12 to 6.58; P = .027) cohorts. Conclusion This multimarker prognostic assay, an independent determinant of melanoma survival, might be beneficial in improving the selection of stage II patients for adjuvant therapy. PMID:19884546

  13. Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy.

    PubMed

    Tam, Frederick W K; Riser, Bruce L; Meeran, Karim; Rambow, JoAnn; Pusey, Charles D; Frankel, Andrew H

    2009-07-01

    Profibrotic growth factors and inflammatory chemokines have been implicated in the pathogenesis of diabetic nephropathy (DN). However, measurement of urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers has not previously been reported, and neither have two such molecules in urine been examined in a single study of DN. In this prospective observational study, 43 adult diabetic patients were studied, 40 were followed up for 6years. Urinary MCP-1/creatinine ratios were found to be significantly higher in patients with macroalbuminuria (3.3- and 2.1-fold higher (p<0.01) than normoalbuminuric and microalbuminuric patients, respectively). CCN2 exhibited a pattern different from that of urinary MCP-1. Urinary CCN2/creatinine ratios were greatly elevated in both microalbuminuric and macroalbuminuric patients (125- and 74-fold higher than normoalbuminuric patients, respectively, p<0.01 and p<0.05, respectively). Further, urinary CCN2, but not MCP-1, correlated with progression of microalbuminuria (R=0.49, p<0.05). In contrast, MCP-1, but not CCN2, correlated with the rate of eGFR decline for all patients (R=0.61, p<0.0001), reflective of its predictive value in patients with macroalbuminuria, but not for patients with microalbuminuria or normoalbuminuria. In conclusion, increased urinary CCN2 is associated with the early progression of DN, whereas MCP-1 is associated with later stage disease. PMID:19409809

  14. A high number of IgG4-positive cells in gastric cancer tissue is associated with tumor progression and poor prognosis.

    PubMed

    Miyatani, Kozo; Saito, Hiroaki; Murakami, Yuki; Watanabe, Joji; Kuroda, Hirohiko; Matsunaga, Tomoyuki; Fukumoto, Yoji; Osaki, Tomohiro; Nakayama, Yuji; Umekita, Yoshihisa; Ikeguchi, Masahide

    2016-05-01

    IgG4-related disease is a newly defined disease characterized by elevated serum IgG4 levels and infiltration of affected organs by IgG4-positive plasma cells. Recently, increased IgG4 levels were reported to be closely related with malignancy. To assess the relationship between IgG4 and the progression of gastric cancer, we immunohistochemically stained in this study gastric cancer tissue samples for IgG4-positive cells using an anti-IgG4 antibody. In addition, pre- and postoperative serum concentrations of IgG4 were measured, using an enzyme-linked immunosorbent assay. In gastric cancer samples, the number of CD138-positive plasma cells was significantly lower and the number of IgG4-positive cells significantly higher than in non-cancerous gastric mucosa. The number of IgG4-positive cells was significantly correlated with gross tumor appearance, tumor depth, lymph node metastasis, venous invasion, and lymphatic invasion. Prognosis was significantly poorer in patients with a high number of IgG4-positive cells than in those with a low number. Multivariate analysis indicated that both the number of IgG4-positive cells and the depth of tumor invasion were independently prognostic of survival. In conclusion, in gastric cancer, the number of IgG4-positive cells is increased and this is closely associated with gastric cancer progression.

  15. Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV.

    PubMed

    Qin, Yannan; Zhong, Yaogang; Ma, Tianran; Wu, Fei; Wu, Haoxiang; Yu, Hanjie; Huang, Chen; Li, Zheng

    2016-04-01

    The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies. PMID:26833199

  16. Comparison of microarray preprocessing methods.

    PubMed

    Shakya, K; Ruskin, H J; Kerr, G; Crane, M; Becker, J

    2010-01-01

    Data preprocessing in microarray technology is a crucial initial step before data analysis is performed. Many preprocessing methods have been proposed but none has proved to be ideal to date. Frequently, datasets are limited by laboratory constraints so that the need is for guidelines on quality and robustness, to inform further experimentation while data are yet restricted. In this paper, we compared the performance of four popular methods, namely MAS5, Li & Wong pmonly (LWPM), Li & Wong subtractMM (LWMM), and Robust Multichip Average (RMA). The comparison is based on the analysis carried out on sets of laboratory-generated data from the Bioinformatics Lab, National Institute of Cellular Biotechnology (NICB), Dublin City University, Ireland. These experiments were designed to examine the effect of Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) treatment in deep lamellar keratoplasty (DLKP) cells. The methodology employed is to assess dispersion across the replicates and analyze the false discovery rate. From the dispersion analysis, we found that variability is reduced more effectively by LWPM and RMA methods. From the false positive analysis, and for both parametric and nonparametric approaches, LWMM is found to perform best. Based on a complementary q-value analysis, LWMM approach again is the strongest candidate. The indications are that, while LWMM is marginally less effective than LWPM and RMA in terms of variance reduction, it has considerably improved discrimination overall.

  17. AMIC@: All MIcroarray Clusterings @ once.

    PubMed

    Geraci, Filippo; Pellegrini, Marco; Renda, M Elena

    2008-07-01

    The AMIC@ Web Server offers a light-weight multi-method clustering engine for microarray gene-expression data. AMIC@ is a highly interactive tool that stresses user-friendliness and robustness by adopting AJAX technology, thus allowing an effective interleaved execution of different clustering algorithms and inspection of results. Among the salient features AMIC@ offers, there are: (i) automatic file format detection, (ii) suggestions on the number of clusters using a variant of the stability-based method of Tibshirani et al. (iii) intuitive visual inspection of the data via heatmaps and (iv) measurements of the clustering quality using cluster homogeneity. Large data sets can be processed efficiently by selecting algorithms (such as FPF-SB and k-Boost), specifically designed for this purpose. In case of very large data sets, the user can opt for a batch-mode use of the system by means of the Clustering wizard that runs all algorithms at once and delivers the results via email. AMIC@ is freely available and open to all users with no login requirement at the following URL http://bioalgo.iit.cnr.it/amica.

  18. 3D Biomaterial Microarrays for Regenerative Medicine: Current State-of-the-Art, Emerging Directions and Future Trends.

    PubMed

    Gaharwar, Akhilesh K; Arpanaei, Ayyoob; Andresen, Thomas L; Dolatshahi-Pirouz, Alireza

    2016-01-27

    Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue-like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem cell differentiation into tissue-specific lineages. The use of 3D biomaterial microarrays can, if optimized correctly, result in a more than 1000-fold reduction in biomaterials and cells consumption when engineering optimal materials combinations, which makes these miniaturized systems very attractive for tissue engineering and drug screening applications.

  19. Protein Microarrays: Novel Developments and Applications

    PubMed Central

    Berrade, Luis; Garcia, Angie E.

    2011-01-01

    Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. They can be used to study most major types of interactions and enzymatic activities that take place in biochemical pathways and have been used for the analysis of simultaneous multiple biomolecular interactions involving protein-protein, protein-lipid, protein-DNA and protein-small molecule interactions. Because of this unique ability to analyze many kinds of molecular interactions en masse, the requirement of very small sample amount and the potential to be miniaturized and automated, protein microarrays are extremely well suited for protein profiling, drug discovery, drug target identification and clinical prognosis and diagnosis. The aim of this review is to summarize the most recent developments in the production, applications and analysis of protein microarrays. PMID:21116694

  20. Quality Visualization of Microarray Datasets Using Circos

    PubMed Central

    Koch, Martin; Wiese, Michael

    2012-01-01

    Quality control and normalization is considered the most important step in the analysis of microarray data. At present there are various methods available for quality assessments of microarray datasets. However there seems to be no standard visualization routine, which also depicts individual microarray quality. Here we present a convenient method for visualizing the results of standard quality control tests using Circos plots. In these plots various quality measurements are drawn in a circular fashion, thus allowing for visualization of the quality and all outliers of each distinct array within a microarray dataset. The proposed method is intended for use with the Affymetrix Human Genome platform (i.e., GPL 96, GPL570 and GPL571). Circos quality measurement plots are a convenient way for the initial quality estimate of Affymetrix datasets that are stored in publicly available databases.

  1. The gene expression signatures of melanoma progression

    PubMed Central

    Haqq, Christopher; Nosrati, Mehdi; Sudilovsky, Daniel; Crothers, Julia; Khodabakhsh, Daniel; Pulliam, Brian L.; Federman, Scot; Miller, James R.; Allen, Robert E.; Singer, Mark I.; Leong, Stanley P. L.; Ljung, Britt-Marie; Sagebiel, Richard W.; Kashani-Sabet, Mohammed

    2005-01-01

    Because of the paucity of available tissue, little information has previously been available regarding the gene expression profiles of primary melanomas. To understand the molecular basis of melanoma progression, we compared the gene expression profiles of a series of nevi, primary melanomas, and melanoma metastases. We found that metastatic melanomas exhibit two dichotomous patterns of gene expression, which unexpectedly reflect gene expression differences already apparent in comparing laser-capture microdissected radial and vertical phases of a large primary melanoma. Unsupervised hierarchical clustering accurately separated nevi and primary melanomas. Multiclass significance analysis of microarrays comparing normal skin, nevi, primary melanomas, and the two types of metastatic melanoma identified 2,602 transcripts that significantly correlated with sample class. These results suggest that melanoma pathogenesis can be understood as a series of distinct molecular events. The gene expression signatures identified here provide the basis for developing new diagnostics and targeting therapies for patients with malignant melanoma. PMID:15833814

  2. Collagenase and tissue plasminogen activator production in developing rat calvariae: normal progression despite fetal exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Davis, B. A.; Sipe, B.; Gershan, L. A.; Fiacco, G. J.; Lorenz, T. C.; Jeffrey, J. J.; Partridge, N. C.

    1998-01-01

    Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence

  3. Contributions to Statistical Problems Related to Microarray Data

    ERIC Educational Resources Information Center

    Hong, Feng

    2009-01-01

    Microarray is a high throughput technology to measure the gene expression. Analysis of microarray data brings many interesting and challenging problems. This thesis consists three studies related to microarray data. First, we propose a Bayesian model for microarray data and use Bayes Factors to identify differentially expressed genes. Second, we…

  4. Photopatterning of Hydrogel Microarrays in Closed Microchips.

    PubMed

    Gumuscu, Burcu; Bomer, Johan G; van den Berg, Albert; Eijkel, Jan C T

    2015-12-14

    To date, optical lithography has been extensively used for in situ patterning of hydrogel structures in a scale range from hundreds of microns to a few millimeters. The two main limitations which prevent smaller feature sizes of hydrogel structures are (1) the upper glass layer of a microchip maintains a large spacing (typically 525 μm) between the photomask and hydrogel precursor, leading to diffraction of UV light at the edges of mask patterns, (2) diffusion of free radicals and monomers results in irregular polymerization near the illumination interface. In this work, we present a simple approach to enable the use of optical lithography to fabricate hydrogel arrays with a minimum feature size of 4 μm inside closed microchips. To achieve this, we combined two different techniques. First, the upper glass layer of the microchip was thinned by mechanical polishing to reduce the spacing between the photomask and hydrogel precursor, and thereby the diffraction of UV light at the edges of mask patterns. The polishing process reduces the upper layer thickness from ∼525 to ∼100 μm, and the mean surface roughness from 20 to 3 nm. Second, we developed an intermittent illumination technique consisting of short illumination periods followed by relatively longer dark periods, which decrease the diffusion of monomers. Combination of these two methods allows for fabrication of 0.4 × 10(6) sub-10 μm sized hydrogel patterns over large areas (cm(2)) with high reproducibility (∼98.5% patterning success). The patterning method is tested with two different types of photopolymerizing hydrogels: polyacrylamide and polyethylene glycol diacrylate. This method enables in situ fabrication of well-defined hydrogel patterns and presents a simple approach to fabricate 3-D hydrogel matrices for biomolecule separation, biosensing, tissue engineering, and immobilized protein microarray applications.

  5. Photopatterning of Hydrogel Microarrays in Closed Microchips.

    PubMed

    Gumuscu, Burcu; Bomer, Johan G; van den Berg, Albert; Eijkel, Jan C T

    2015-12-14

    To date, optical lithography has been extensively used for in situ patterning of hydrogel structures in a scale range from hundreds of microns to a few millimeters. The two main limitations which prevent smaller feature sizes of hydrogel structures are (1) the upper glass layer of a microchip maintains a large spacing (typically 525 μm) between the photomask and hydrogel precursor, leading to diffraction of UV light at the edges of mask patterns, (2) diffusion of free radicals and monomers results in irregular polymerization near the illumination interface. In this work, we present a simple approach to enable the use of optical lithography to fabricate hydrogel arrays with a minimum feature size of 4 μm inside closed microchips. To achieve this, we combined two different techniques. First, the upper glass layer of the microchip was thinned by mechanical polishing to reduce the spacing between the photomask and hydrogel precursor, and thereby the diffraction of UV light at the edges of mask patterns. The polishing process reduces the upper layer thickness from ∼525 to ∼100 μm, and the mean surface roughness from 20 to 3 nm. Second, we developed an intermittent illumination technique consisting of short illumination periods followed by relatively longer dark periods, which decrease the diffusion of monomers. Combination of these two methods allows for fabrication of 0.4 × 10(6) sub-10 μm sized hydrogel patterns over large areas (cm(2)) with high reproducibility (∼98.5% patterning success). The patterning method is tested with two different types of photopolymerizing hydrogels: polyacrylamide and polyethylene glycol diacrylate. This method enables in situ fabrication of well-defined hydrogel patterns and presents a simple approach to fabricate 3-D hydrogel matrices for biomolecule separation, biosensing, tissue engineering, and immobilized protein microarray applications. PMID:26558488

  6. The Impact of Photobleaching on Microarray Analysis.

    PubMed

    von der Haar, Marcel; Preuß, John-Alexander; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2015-01-01

    DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner's laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube's voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results. PMID:26378589

  7. Automated analytical microarrays: a critical review.

    PubMed

    Seidel, Michael; Niessner, Reinhard

    2008-07-01

    Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications.

  8. Evaluation of Surface Chemistries for Antibody Microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; White, Amanda M.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-12-01

    Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been primarily derived from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but no rigorous studies that compare these different surfaces. Therefore, we have used an enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 16 different commercially available slide types. Full standard curves were generated for 24 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types can produce useful data, glass slides coated with poly-L-lysine or aminosilane, with or without activation with a crosslinker, consistently produce superior results in the ELISA microarray analyses we performed.

  9. 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.

  10. Microarray technology to investigate genes associated with papillary thyroid carcinoma.

    PubMed

    Zhu, Xinyong; Yao, Jing; Tian, Wen

    2015-05-01

    DNA microarray data on thyroid tissue from patients with papillary thyroid carcinoma (PTC) and from healthy controls were compared in order to investigate the regulatory genes and uncover the underlying regulatory network in PTC. The DNA microarray data set, GSE3678, was downloaded from Gene Expression Omnibus database. This included seven thyroid tissue samples from patients with PTC and seven samples from healthy controls. Raw data were processed and differentially expressed genes (DEGs) were identified using corresponding R packages. Gene regulation analysis was conducted using TRANSFAC® and TRED. A total of 171 DEGs were obtained. A regulatory network was then established, using 104 of the DEGs. Subsequently, pathway enrichment analyses of the genes were conducted using Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool. Three differentially expressed transcription factors were identified: Trefoil factor 3, cut‑like homeobox 2 and forkhead box protein A2. The most significant pathways involving the 104 DEGs were pathways involved in cancer. Biological process analysis using DAVID, suggested that these genes were associated with the positive regulation of gene expression, gene transcription and metabolic processes. The present study identified a range of genes associated with the development of PTC. The results of the present study were beneficial for understanding the regulatory mechanisms involved in PTC, and for developing clinical diagnostic and therapeutic approaches for this disease.

  11. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

    PubMed Central

    Delalat, Bahman; Rojas-Canales, Darling M.; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J.; Penko, Daniella; Drogemuller, Christopher J.; Loudovaris, Thomas; Coates, Patrick T. H.; Voelcker, Nicolas H.

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  12. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

    PubMed

    Delalat, Bahman; Rojas-Canales, Darling M; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J; Penko, Daniella; Drogemuller, Christopher J; Loudovaris, Thomas; Coates, Patrick T H; Voelcker, Nicolas H

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  13. Assembly of ordered microsphere arrays: Platforms for microarrays

    NASA Astrophysics Data System (ADS)

    Xu, Wanling

    Microarrays are powerful tools in gene expression assessment, protein profiling, and protein function screening, as well as cell and tissue analysis. With thousands of small array spots assembled in an ordered array, these small devices makes it possible to screen for multiple targets in a fast, parallel, high-throughput manner. The well-developed technology of DNA microarrays, also called DNA chips, has proved successful in all kinds of biological experiments, including the human genome-sequencing project. The development of protein arrays has lagged behind that of DNA arrays mainly because of the greater complexity of proteins. Some parts of the microarray technology can be transplanted into the realm of protein arrays, while others cannot. The challenges from the complexity of protein targets demand more robust and powerful devices. Traditional planar arrays, in which proteins bind directly to a planar surface, have a drawback in that some proteins will be denatured or cluster together after immobilization. Microsphere-based microarrays represent a more advanced strategy. The functional proteins are first attached to microspheres; these microspheres are then immobilized in arrays on a planar surface. In this dissertation, two approaches to assembling arrays of microspheres will be discussed. The hydrodynamic approach uses surface micromachining and Deep Reactive Ion Etching techniques to form an array of channels through a silicon wafer. By drawing fluid containing the microspheres through the channels they become trapped in the channels and thereby immobilized. In the magnetic approach, permalloy films are deposited on a silicon substrate and subsequently patterned to form magnetic attachment sites. An external magnetic field is then applied and the magnetic microspheres then assemble on these sites. Both devices are able to immobilize microspheres in an ordered array, as opposed to coarsely grouping them in array spots. The assembled arrays are robust in that

  14. Single nucleotide polymorphism-based microarray analysis for the diagnosis of hydatidiform moles

    PubMed Central

    XIE, YINGJUN; PEI, XIAOJUAN; DONG, YU; WU, HUIQUN; WU, JIANZHU; SHI, HUIJUAN; ZHUANG, XUYING; SUN, XIAOFANG; HE, JIALING

    2016-01-01

    In clinical diagnostics, single nucleotide polymorphism (SNP)-based microarray analysis enables the detection of copy number variations (CNVs), as well as copy number neutral regions, that are absent of heterozygosity throughout the genome. The aim of the present study was to evaluate the effectiveness and sensitivity of SNP-based microarray analysis in the diagnosis of hydatidiform mole (HM). By using whole-genome SNP microarray analysis, villous genotypes were detected, and the ploidy of villous tissue was determined to identify HMs. A total of 66 villous tissues and two twin tissues were assessed in the present study. Among these samples, 11 were triploid, one was tetraploid, 23 were abnormal aneuploidy, three were complete genome homozygosity, and the remaining ones were normal ploidy. The most noteworthy finding of the present study was the identification of six partial HMs and three complete HMs from those samples that were not identified as being HMs on the basis of the initial diagnosis of experienced obstetricians. This study has demonstrated that the application of an SNP-based microarray analysis was able to increase the sensitivity of diagnosis for HMs with partial and complete HMs, which makes the identification of these diseases at an early gestational age possible. PMID:27151252

  15. Empirical Bayes ranking and selection methods via semiparametric hierarchical mixture models in microarray studies.

    PubMed

    Noma, Hisashi; Matsui, Shigeyuki

    2013-05-20

    The main purpose of microarray studies is screening of differentially expressed genes as candidates for further investigation. Because of limited resources in this stage, prioritizing genes are relevant statistical tasks in microarray studies. For effective gene selections, parametric empirical Bayes methods for ranking and selection of genes with largest effect sizes have been proposed (Noma et al., 2010; Biostatistics 11: 281-289). The hierarchical mixture model incorporates the differential and non-differential components and allows information borrowing across differential genes with separation from nuisance, non-differential genes. In this article, we develop empirical Bayes ranking methods via a semiparametric hierarchical mixture model. A nonparametric prior distribution, rather than parametric prior distributions, for effect sizes is specified and estimated using the "smoothing by roughening" approach of Laird and Louis (1991; Computational statistics and data analysis 12: 27-37). We present applications to childhood and infant leukemia clinical studies with microarrays for exploring genes related to prognosis or disease progression.

  16. Microarrays: molecular allergology and nanotechnology for personalised medicine (I).

    PubMed

    Lucas, J M

    2010-01-01

    The diagnosis of antibody-mediated allergic disorders is based on the clinical findings and the detection of allergen-specific IgE based on in vitro and in vivo techniques, together with allergen provocation tests. In vitro diagnostic techniques have progressed enormously following the introduction of the advances made in proteomics and nanotechnology--offering tools for the diagnosis and investigation of allergy at molecular level. The most advanced developments are the microarray techniques, which in genomics allowed rapid description of the human genetic code, and which now have been applied to proteomics, broadening the field for research and clinical use. Together with these technological advances, the characterisation of most of the different proteins generating specific IgE and which conform each natural allergen, as well as their purification or genetic engineering-based synthesis, have been crucial elements--offering the possibility of identifying disease-causing allergens at molecular level, establishing a component-resolved diagnosis (CRD), using them to study the natural course of the disease, and applying them to improvements in specific immunotherapy. Microarrays of allergic components offer results relating to hundreds of these allergenic components in a single test, and use a small amount of serum that can be obtained from capillary blood. The availability of new molecules will allow the development of panels including new allergenic components and sources, which will require evaluation for clinical use. The present study reviews these new developments, component-resolved diagnosis, and the development of microarray techniques as a critical element for furthering our knowledge of allergic disease.

  17. DNA microarray analyses in higher plants.

    PubMed

    Galbraith, David W

    2006-01-01

    DNA microarrays were originally devised and described as a convenient technology for the global analysis of plant gene expression. Over the past decade, their use has expanded enormously to cover all kingdoms of living organisms. At the same time, the scope of applications of microarrays has increased beyond expression analyses, with plant genomics playing a leadership role in the on-going development of this technology. As the field has matured, the rate-limiting step has moved from that of the technical process of data generation to that of data analysis. We currently face major problems in dealing with the accumulating datasets, not simply with respect to how to archive, access, and process the huge amounts of data that have been and are being produced, but also in determining the relative quality of the different datasets. A major recognized concern is the appropriate use of statistical design in microarray experiments, without which the datasets are rendered useless. A vigorous area of current research involves the development of novel statistical tools specifically for microarray experiments. This article describes, in a necessarily selective manner, the types of platforms currently employed in microarray research and provides an overview of recent activities using these platforms in plant biology.

  18. Oligonucleotide microarrays in constitutional genetic diagnosis.

    PubMed

    Keren, Boris; Le Caignec, Cedric

    2011-06-01

    Oligonucleotide microarrays such as comparative genomic hybridization arrays and SNP microarrays enable the identification of genomic imbalances - also termed copy-number variants - with increasing resolution. This article will focus on the most significant applications of high-throughput oligonucleotide microarrays, both in genetic diagnosis and research. In genetic diagnosis, the method is becoming a standard tool for investigating patients with unexplained developmental delay/intellectual disability, autism spectrum disorders and/or with multiple congenital anomalies. Oligonucleotide microarray have also been recently applied to the detection of genomic imbalances in prenatal diagnosis either to characterize a chromosomal rearrangement that has previously been identified by standard prenatal karyotyping or to detect a cryptic genomic imbalance in a fetus with ultrasound abnormalities and a normal standard prenatal karyotype. In research, oligonucleotide microarrays have been used for a wide range of applications, such as the identification of new genes responsible for monogenic disorders and the association of a copy-number variant as a predisposing factor to a common disease. Despite its widespread use, the interpretation of results is not always straightforward. We will discuss several unexpected results and ethical issues raised by these new methods.

  19. Advancing Microarray Assembly with Acoustic Dispensing Technology

    PubMed Central

    Wong, E. Y.; Diamond, S. L.

    2011-01-01

    In the assembly of microarrays and microarray-based chemical assays and enzymatic bioassays, most approaches use pins for contact spotting. Acoustic dispensing is a technology capable of nanoliter transfers by using acoustic energy to eject liquid sample from an open source well. Although typically used for well plate transfers, when applied to microarraying it avoids drawbacks of undesired physical contact with sample, difficulty in assembling multicomponent reactions on a chip by readdressing, a rigid mode of printing that lacks patterning capabilities, and time-consuming wash steps. We demonstrated the utility of acoustic dispensing by delivering human cathepsin L in a drop-on-drop fashion into individual 50-nanoliter, pre-spotted reaction volumes to activate enzyme reactions at targeted positions on a microarray. We generated variable-sized spots ranging from 200 to 750 μm (and higher), and handled the transfer of fluorescent bead suspensions with increasing source well concentrations of 0.1 to 10 ×108 beads/mL in a linear fashion. There are no tips that can clog and liquid dispensing CVs are generally below 5%. This platform expands the toolbox for generating analytical arrays and meets needs associated with spatially-addressed assembly of multicomponent microarrays on the nanoliter scale. PMID:19035650

  20. A Synthetic Kinome Microarray Data Generator

    PubMed Central

    Maleki, Farhad; Kusalik, Anthony

    2015-01-01

    Cellular pathways involve the phosphorylation and dephosphorylation of proteins. Peptide microarrays called kinome arrays facilitate the measurement of the phosphorylation activity of hundreds of proteins in a single experiment. Analyzing the data from kinome microarrays is a multi-step process. Typically, various techniques are possible for a particular step, and it is necessary to compare and evaluate them. Such evaluations require data for which correct analysis results are known. Unfortunately, such kinome data is not readily available in the community. Further, there are no established techniques for creating artificial kinome datasets with known results and with the same characteristics as real kinome datasets. In this paper, a methodology for generating synthetic kinome array data is proposed. The methodology relies on actual intensity measurements from kinome microarray experiments and preserves their subtle characteristics. The utility of the methodology is demonstrated by evaluating methods for eliminating heterogeneous variance in kinome microarray data. Phosphorylation intensities from kinome microarrays often exhibit such heterogeneous variance and its presence can negatively impact downstream statistical techniques that rely on homogeneity of variance. It is shown that using the output from the proposed synthetic data generator, it is possible to critically compare two variance stabilization methods. PMID:27600233

  1. Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells

    PubMed Central

    Dittmar, Thomas; Zänker, Kurt S.

    2015-01-01

    The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that “accidental” tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells. PMID:26703575

  2. Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways

    PubMed Central

    Valle, C; Salvatori, I; Gerbino, V; Rossi, S; Palamiuc, L; René, F; Carrì, M T

    2014-01-01

    Acetylation homeostasis is thought to play a role in amyotrophic lateral sclerosis, and treatment with inhibitors of histone deacetylases has been considered a potential and attractive therapeutic approach, despite the lack of a thorough study of this class of proteins. In this study, we have considerably extended previous knowledge on the expression of 13 histone deacetylases in tissues (spinal cord and muscle) from mice carrying two different ALS-linked SOD1 mutations (G93A-SOD1 and G86R-SOD1). We have then focused on class III histone deacetylases SIRT1 and SIRT2 that are considered relevant in neurodegenerative diseases. SIRT1 decreases in the spinal cord, but increases in muscle during the progression of the disease, and a similar expression pattern is observed in the corresponding cell models (neuroblastoma and myoblasts). SIRT2 mRNA expression increases in the spinal cord in both G93A-SOD1 and G86R-SOD1 mice but protein expression is substantially unchanged in all the models examined. At variance with other sirtuin modulators (sirtinol, AGK2 and SRT1720), the well-known SIRT1 inhibitor Ex527 has positive effects on survival of neuronal cells expressing mutant SOD1, but this effect is neither mediated by SIRT1 inhibition nor by SIRT2 inhibition. These data call for caution in proposing sirtuin modulation as a target for treatment. PMID:24946089

  3. Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients

    PubMed Central

    Wang, Chih-Yang; Lai, Ming-Derg; Phan, Nam Nhut; Sun, Zhengda; Lin, Yen-Chang

    2015-01-01

    Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment. PMID:26147197

  4. Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients.

    PubMed

    Wang, Chih-Yang; Lai, Ming-Derg; Phan, Nam Nhut; Sun, Zhengda; Lin, Yen-Chang

    2015-01-01

    Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment.

  5. Identification of Novel Cholesteatoma-Related Gene Expression Signatures Using Full-Genome Microarrays

    PubMed Central

    Klenke, Christin; Janowski, Sebastian; Borck, Daniela; Widera, Darius; Ebmeyer, Jörg; Kalinowski, Jörn; Leichtle, Anke; Hofestädt, Ralf; Upile, Tahwinder; Kaltschmidt, Christian; Kaltschmidt, Barbara; Sudhoff, Holger

    2012-01-01

    Background Cholesteatoma is a gradually expanding destructive epithelial lesion within the middle ear. It can cause extensive local tissue destruction in the temporal bone and can initially lead to the development of conductive hearing loss via ossicular erosion. As the disease progresses, sensorineural hearing loss, vertigo or facial palsy may occur. Cholesteatoma may promote the spread of infection through the tegmen of the middle ear and cause meningitis or intracranial infections with abscess formation. It must, therefore, be considered as a potentially life-threatening middle ear disease. Methods and Findings In this study, we investigated differentially expressed genes in human cholesteatomas in comparison to regular auditory canal skin using Whole Human Genome Microarrays containing 19,596 human genes. In addition to already described up-regulated mRNAs in cholesteatoma, such as MMP9, DEFB2 and KRT19, we identified 3558 new cholesteatoma-related transcripts. 811 genes appear to be significantly differentially up-regulated in cholesteatoma. 334 genes were down-regulated more than 2-fold. Significantly regulated genes with protein metabolism activity include matrix metalloproteinases as well as PI3, SERPINB3 and SERPINB4. Genes like SPP1, KRT6B, PRPH, SPRR1B and LAMC2 are known as genes with cell growth and/or maintenance activity. Transport activity genes and signal transduction genes are LCN2, GJB2 and CEACAM6. Three cell communication genes were identified; one CDH19 and two from the S100 family. Conclusions This study demonstrates that the expression profile of cholesteatoma is similar to a metastatic tumour and chronically inflamed tissue. Based on the investigated profiles we present novel protein-protein interaction and signal transduction networks, which include cholesteatoma-regulated transcripts and may be of great value for drug targeting and therapy development. PMID:23285167

  6. Protein microarrays for parasite antigen discovery.

    PubMed

    Driguez, Patrick; Doolan, Denise L; Molina, Douglas M; Loukas, Alex; Trieu, Angela; Felgner, Phil L; McManus, Donald P

    2015-01-01

    The host serological profile to a parasitic infection, such as schistosomiasis, can be used to define potential vaccine and diagnostic targets. Determining the host antibody response using traditional approaches is hindered by the large number of putative antigens in any parasite proteome. Parasite protein microarrays offer the potential for a high-throughput host antibody screen to simplify this task. In order to construct the array, parasite proteins are selected from available genomic sequence and protein databases using bioinformatic tools. Selected open reading frames are PCR amplified, incorporated into a vector for cell-free protein expression, and printed robotically onto glass slides. The protein microarrays can be probed with antisera from infected/immune animals or humans and the antibody reactivity measured with fluorophore labeled antibodies on a confocal laser microarray scanner to identify potential targets for diagnosis or therapeutic or prophylactic intervention. PMID:25388117

  7. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  8. The use of microarrays in microbial ecology

    SciTech Connect

    Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L.; Zhou, J.

    2009-09-15

    Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer

  9. Analyzing Schizophrenia by DNA Microarrays

    PubMed Central

    Horváth, Szatmár; Janka, Zoltán; Mirnics, Károly

    2010-01-01

    To understand the pathological processes of schizophrenia we must embrace the analysis of the diseased human brain: we will never be able to recapitulate the pathology of uniquely human disorders in an animal model. Based on the outcome of the transcriptome profiling experiments performed to date it appears that schizophrenia is associated with a global gene expression disturbance across many cortical regions. In addition, transcriptome changes are present in multiple cell types, including specific subclasses of principal neurons, interneurons and oligodendrocytes. Furthermore, transcripts related to synaptic transmission, energy metabolism and inhibitory neurotransmission are routinely found underexpressed in the postmortem brain tissue of subjects with schizophrenia. To put these transcriptome data in biological context we must make our data publicly available and report our findings in a proper, expanded MIAME format. Cell type specific expression profiling and sequencing-based transcripts assessments should be expanded, with particular attention to understanding splice-variant changes in various mental disorders. Deciphering the pathophysiology of mental disorders depends on integrating data from across many research fields and techniques. Leads from postmortem transcriptome profiling will be essential to generate model animals, perform tissue culture experiments and develop or evaluate novel drugs to treat this devastating disorder. PMID:20801428

  10. Preparation and use of reverse protein microarrays.

    PubMed

    Pin, Elisa; Federici, Giulia; Petricoin, Emanuel F

    2014-01-01

    Reverse-phase protein array (RPPA) is a multiplex, high-throughput proteomic technique for profiling the activation status of signal transduction pathways involved in cancer survival and progression, potentially allowing for identification of new biomarkers and drug targets. On RPPA, the entire patient proteome is immobilized on a spot and single proteins can be quantified across a set of samples, spotted on the same array, with high specificity and sensitivity. Array immunostaining and signal amplification systems are used to generate a signal proportional to the concentration of the analyte. Dedicated scanners and software are used to detect spots, measure intensity, subtract background, normalize signal, and generate a numeric value as output. The generated output file is then analyzed using several different bioinformatic and biostatistical tools. In this unit, the RPPA procedure is described in depth, from sample handling and preparation to data analysis, with particular emphasis on tissue sample analysis. PMID:24510676

  11. Micro-Analyzer: automatic preprocessing of Affymetrix microarray data.

    PubMed

    Guzzi, Pietro Hiram; Cannataro, Mario

    2013-08-01

    A current trend in genomics is the investigation of the cell mechanism using different technologies, in order to explain the relationship among genes, molecular processes and diseases. For instance, the combined use of gene-expression arrays and genomic arrays has been demonstrated as an effective instrument in clinical practice. Consequently, in a single experiment different kind of microarrays may be used, resulting in the production of different types of binary data (images and textual raw data). The analysis of microarray data requires an initial preprocessing phase, that makes raw data suitable for use on existing analysis platforms, such as the TIGR M4 (TM4) Suite. An additional challenge to be faced by emerging data analysis platforms is the ability to treat in a combined way those different microarray formats coupled with clinical data. In fact, resulting integrated data may include both numerical and symbolic data (e.g. gene expression and SNPs regarding molecular data), as well as temporal data (e.g. the response to a drug, time to progression and survival rate), regarding clinical data. Raw data preprocessing is a crucial step in analysis but is often performed in a manual and error prone way using different software tools. Thus novel, platform independent, and possibly open source tools enabling the semi-automatic preprocessing and annotation of different microarray data are needed. The paper presents Micro-Analyzer (Microarray Analyzer), a cross-platform tool for the automatic normalization, summarization and annotation of Affymetrix gene expression and SNP binary data. It represents the evolution of the μ-CS tool, extending the preprocessing to SNP arrays that were not allowed in μ-CS. The Micro-Analyzer is provided as a Java standalone tool and enables users to read, preprocess and analyse binary microarray data (gene expression and SNPs) by invoking TM4 platform. It avoids: (i) the manual invocation of external tools (e.g. the Affymetrix Power

  12. Micro-Analyzer: automatic preprocessing of Affymetrix microarray data.

    PubMed

    Guzzi, Pietro Hiram; Cannataro, Mario

    2013-08-01

    A current trend in genomics is the investigation of the cell mechanism using different technologies, in order to explain the relationship among genes, molecular processes and diseases. For instance, the combined use of gene-expression arrays and genomic arrays has been demonstrated as an effective instrument in clinical practice. Consequently, in a single experiment different kind of microarrays may be used, resulting in the production of different types of binary data (images and textual raw data). The analysis of microarray data requires an initial preprocessing phase, that makes raw data suitable for use on existing analysis platforms, such as the TIGR M4 (TM4) Suite. An additional challenge to be faced by emerging data analysis platforms is the ability to treat in a combined way those different microarray formats coupled with clinical data. In fact, resulting integrated data may include both numerical and symbolic data (e.g. gene expression and SNPs regarding molecular data), as well as temporal data (e.g. the response to a drug, time to progression and survival rate), regarding clinical data. Raw data preprocessing is a crucial step in analysis but is often performed in a manual and error prone way using different software tools. Thus novel, platform independent, and possibly open source tools enabling the semi-automatic preprocessing and annotation of different microarray data are needed. The paper presents Micro-Analyzer (Microarray Analyzer), a cross-platform tool for the automatic normalization, summarization and annotation of Affymetrix gene expression and SNP binary data. It represents the evolution of the μ-CS tool, extending the preprocessing to SNP arrays that were not allowed in μ-CS. The Micro-Analyzer is provided as a Java standalone tool and enables users to read, preprocess and analyse binary microarray data (gene expression and SNPs) by invoking TM4 platform. It avoids: (i) the manual invocation of external tools (e.g. the Affymetrix Power

  13. Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

    PubMed

    Ordóñez, Raquel; Gallo-Oller, Gabriel; Martínez-Soto, Soledad; Legarra, Sheila; Pata-Merci, Noémie; Guegan, Justine; Danglot, Giselle; Bernheim, Alain; Meléndez, Bárbara; Rey, Juan A; Castresana, Javier S

    2014-01-01

    Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells. PMID:25392930

  14. A new method for gridding DNA microarrays.

    PubMed

    Charalambous, Christoforos C; Matsopoulos, George K

    2013-10-01

    In this paper, a new methodological scheme for the gridding of DNA microarrays is proposed. The scheme composes of a series of processes applied sequentially. Each DNA microarray image is pre-processed to remove any noise and the center of each spot is detected using a template matching algorithm. Then, an initial gridding is automatically placed on the DNA microarray image by 'building' rectangular pyramids around the detected spots' centers. The gridlines "move" between the pyramids, horizontally and vertically, forming this initial grid. Furthermore, a refinement process is applied composing of a five-step approach in order to correct gridding imperfections caused by its initial placement, both in non-spot cases and in more than one spot enclosure cases. The proposed gridding scheme is applied on DNA microarray images under known transformations and on real-world DNA data. Its performance is compared against the projection pursuit method, which is often used due to its speed and simplicity, as well as against a state-of-the-art method, the Optimal Multi-level Thresholding Gridding (OMTG). According to the obtained results, the proposed gridding scheme outperforms both methods, qualitatively and quantitatively.

  15. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    SciTech Connect

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S.; Golova, Julia; Perov, Alexander N.; Protic, Miroslava; Robison, Richard; Shipma, Matthew; White, Amanda M.; Willse, Alan R.

    2006-01-01

    A diagnostic, genome-independent microbial fingerprinting method using DNA oligonucleotide microarrays was used for high-resolution differentiation between closely related Bacillus strains, including two strains of Bacillus anthracis that are monomorphic (indistinguishable) via amplified fragment length polymorphism fingerprinting techniques. Replicated hybridizations on 391-probe nonamer arrays were used to construct a prototype fingerprint library for quantitative comparisons. Descriptive analysis of the fingerprints, including phylogenetic reconstruction, is consistent with previous taxonomic organization of the genus. Newly developed statistical analysis methods were used to quantitatively compare and objectively confirm apparent differences in microarray fingerprints with the statistical rigor required for microbial forensics and clinical diagnostics. These data suggest that a relatively simple fingerprinting microarray and statistical analysis method can differentiate between species in the Bacillus cereus complex, and between strains of B. anthracis. A synthetic DNA standard was used to understand underlying microarray and process-level variability, leading to specific recommendations for the development of a standard operating procedure and/or continued technology enhancements for microbial forensics and diagnostics.

  16. Data Analysis Strategies for Protein Microarrays

    PubMed Central

    Díez, Paula; Dasilva, Noelia; González-González, María; Matarraz, Sergio; Casado-Vela, Juan; Orfao, Alberto; Fuentes, Manuel

    2012-01-01

    Microarrays constitute a new platform which allows the discovery and characterization of proteins. According to different features, such as content, surface or detection system, there are many types of protein microarrays which can be applied for the identification of disease biomarkers and the characterization of protein expression patterns. However, the analysis and interpretation of the amount of information generated by microarrays remain a challenge. Further data analysis strategies are essential to obtain representative and reproducible results. Therefore, the experimental design is key, since the number of samples and dyes, among others aspects, would define the appropriate analysis method to be used. In this sense, several algorithms have been proposed so far to overcome analytical difficulties derived from fluorescence overlapping and/or background noise. Each kind of microarray is developed to fulfill a specific purpose. Therefore, the selection of appropriate analytical and data analysis strategies is crucial to achieve successful biological conclusions. In the present review, we focus on current algorithms and main strategies for data interpretation.

  17. Shrinkage covariance matrix approach for microarray data

    NASA Astrophysics Data System (ADS)

    Karjanto, Suryaefiza; Aripin, Rasimah

    2013-04-01

    Microarray technology was developed for the purpose of monitoring the expression levels of thousands of genes. A microarray data set typically consists of tens of thousands of genes (variables) from just dozens of samples due to various constraints including the high cost of producing microarray chips. As a result, the widely used standard covariance estimator is not appropriate for this purpose. One such technique is the Hotelling's T2 statistic which is a multivariate test statistic for comparing means between two groups. It requires that the number of observations (n) exceeds the number of genes (p) in the set but in microarray studies it is common that n < p. This leads to a biased estimate of the covariance matrix. In this study, the Hotelling's T2 statistic with the shrinkage approach is proposed to estimate the covariance matrix for testing differential gene expression. The performance of this approach is then compared with other commonly used multivariate tests using a widely analysed diabetes data set as illustrations. The results across the methods are consistent, implying that this approach provides an alternative to existing techniques.

  18. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The primary…

  19. Data Analysis Strategies for Protein Microarrays

    PubMed Central

    Díez, Paula; Dasilva, Noelia; González-González, María; Matarraz, Sergio; Casado-Vela, Juan; Orfao, Alberto; Fuentes, Manuel

    2012-01-01

    Microarrays constitute a new platform which allows the discovery and characterization of proteins. According to different features, such as content, surface or detection system, there are many types of protein microarrays which can be applied for the identification of disease biomarkers and the characterization of protein expression patterns. However, the analysis and interpretation of the amount of information generated by microarrays remain a challenge. Further data analysis strategies are essential to obtain representative and reproducible results. Therefore, the experimental design is key, since the number of samples and dyes, among others aspects, would define the appropriate analysis method to be used. In this sense, several algorithms have been proposed so far to overcome analytical difficulties derived from fluorescence overlapping and/or background noise. Each kind of microarray is developed to fulfill a specific purpose. Therefore, the selection of appropriate analytical and data analysis strategies is crucial to achieve successful biological conclusions. In the present review, we focus on current algorithms and main strategies for data interpretation. PMID:27605336

  20. DISC-BASED IMMUNOASSAY MICROARRAYS. (R825433)

    EPA Science Inventory

    Microarray technology as applied to areas that include genomics, diagnostics, environmental, and drug discovery, is an interesting research topic for which different chip-based devices have been developed. As an alternative, we have explored the principle of compact disc-based...

  1. MICROARRAY DATA ANALYSIS USING MULTIPLE STATISTICAL MODELS

    EPA Science Inventory

    Microarray Data Analysis Using Multiple Statistical Models

    Wenjun Bao1, Judith E. Schmid1, Amber K. Goetz1, Ming Ouyang2, William J. Welsh2,Andrew I. Brooks3,4, ChiYi Chu3,Mitsunori Ogihara3,4, Yinhe Cheng5, David J. Dix1. 1National Health and Environmental Effects Researc...

  2. Raman-based microarray readout: a review.

    PubMed

    Haisch, Christoph

    2016-07-01

    For a quarter of a century, microarrays have been part of the routine analytical toolbox. Label-based fluorescence detection is still the commonest optical readout strategy. Since the 1990s, a continuously increasing number of label-based as well as label-free experiments on Raman-based microarray readout concepts have been reported. This review summarizes the possible concepts and methods and their advantages and challenges. A common label-based strategy is based on the binding of selective receptors as well as Raman reporter molecules to plasmonic nanoparticles in a sandwich immunoassay, which results in surface-enhanced Raman scattering signals of the reporter molecule. Alternatively, capture of the analytes can be performed by receptors on a microarray surface. Addition of plasmonic nanoparticles again leads to a surface-enhanced Raman scattering signal, not of a label but directly of the analyte. This approach is mostly proposed for bacteria and cell detection. However, although many promising readout strategies have been discussed in numerous publications, rarely have any of them made the step from proof of concept to a practical application, let alone routine use. Graphical Abstract Possible realization of a SERS (Surface-Enhanced Raman Scattering) system for microarray readout. PMID:26973235

  3. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  4. Examining microarray slide quality for the EPA using SNL's hyperspectral microarray scanner.

    SciTech Connect

    Rohde, Rachel M.; Timlin, Jerilyn Ann

    2005-11-01

    This report summarizes research performed at Sandia National Laboratories (SNL) in collaboration with the Environmental Protection Agency (EPA) to assess microarray quality on arrays from two platforms of interest to the EPA. Custom microarrays from two novel, commercially produced array platforms were imaged with SNL's unique hyperspectral imaging technology and multivariate data analysis was performed to investigate sources of emission on the arrays. No extraneous sources of emission were evident in any of the array areas scanned. This led to the conclusions that either of these array platforms could produce high quality, reliable microarray data for the EPA toxicology programs. Hyperspectral imaging results are presented and recommendations for microarray analyses using these platforms are detailed within the report.

  5. Identifying Fishes through DNA Barcodes and Microarrays

    PubMed Central

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N.; Weber, Hannes; Blohm, Dietmar

    2010-01-01

    Background International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products. PMID

  6. Facilitating functional annotation of chicken microarray data

    PubMed Central

    2009-01-01

    Background Modeling results from chicken microarray studies is challenging for researchers due to little functional annotation associated with these arrays. The Affymetrix GenChip chicken genome array, one of the biggest arrays that serve as a key research tool for the study of chicken functional genomics, is among the few arrays that link gene products to Gene Ontology (GO). However the GO annotation data presented by Affymetrix is incomplete, for example, they do not show references linked to manually annotated functions. In addition, there is no tool that facilitates microarray researchers to directly retrieve functional annotations for their datasets from the annotated arrays. This costs researchers amount of time in searching multiple GO databases for functional information. Results We have improved the breadth of functional annotations of the gene products associated with probesets on the Affymetrix chicken genome array by 45% and the quality of annotation by 14%. We have also identified the most significant diseases and disorders, different types of genes, and known drug targets represented on Affymetrix chicken genome array. To facilitate functional annotation of other arrays and microarray experimental datasets we developed an Array GO Mapper (AGOM) tool to help researchers to quickly retrieve corresponding functional information for their dataset. Conclusion Results from this study will directly facilitate annotation of other chicken arrays and microarray experimental datasets. Researchers will be able to quickly model their microarray dataset into more reliable biological functional information by using AGOM tool. The disease, disorders, gene types and drug targets revealed in the study will allow researchers to learn more about how genes function in complex biological systems and may lead to new drug discovery and development of therapies. The GO annotation data generated will be available for public use via AgBase website and will be updated on regular

  7. Microarray analysis at single molecule resolution

    PubMed Central

    Mureşan, Leila; Jacak, Jarosław; Klement, Erich Peter; Hesse, Jan; Schütz, Gerhard J.

    2010-01-01

    Bioanalytical chip-based assays have been enormously improved in sensitivity in the recent years; detection of trace amounts of substances down to the level of individual fluorescent molecules has become state of the art technology. The impact of such detection methods, however, has yet not fully been exploited, mainly due to a lack in appropriate mathematical tools for robust data analysis. One particular example relates to the analysis of microarray data. While classical microarray analysis works at resolutions of two to 20 micrometers and quantifies the abundance of target molecules by determining average pixel intensities, a novel high resolution approach [1] directly visualizes individual bound molecules as diffraction limited peaks. The now possible quantification via counting is less susceptible to labeling artifacts and background noise. We have developed an approach for the analysis of high-resolution microarray images. It consists first of a single molecule detection step, based on undecimated wavelet transforms, and second, of a spot identification step via spatial statistics approach (corresponding to the segmentation step in the classical microarray analysis). The detection method was tested on simulated images with a concentration range of 0.001 to 0.5 molecules per square micron and signal-to-noise ratio (SNR) between 0.9 and 31.6. For SNR above 15 the false negatives relative error was below 15%. Separation of foreground/background proved reliable, in case foreground density exceeds background by a factor of 2. The method has also been applied to real data from high-resolution microarray measurements. PMID:20123580

  8. 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

  9. Use of microarray technology to profile gene expression patterns important for reproduction in cattle.

    PubMed

    Evans, A C O; Forde, N; O'Gorman, G M; Zielak, A E; Lonergan, P; Fair, T

    2008-07-01

    Fertility in cattle is a major component of many agricultural enterprises and there is pressure to devise methods to improve this. A number of approaches are ongoing, one of which is to better understand the cellular and molecular events of the development of reproductive tissues and to use these as targets for developing new strategies. Microarray technologies now allow us the potential to determine the transcriptional profile of expressed genes in a given tissue. This review focuses on the types of microarrays available for studies in cattle and concludes that genes associated with one or more of the cellular processes of cell survival/death, intracellular signalling, transcription and translation, cell division and proliferation and cellular metabolism are the main transcriptional pathways that control the development of ovarian follicles, oocytes, early embryos and the uterine endometrium about the time of the establishment of pregnancy.

  10. Ribosomal RNA depletion or exclusion has negligible effect on the detection of viruses in a pan viral microarray.

    PubMed

    McGowan, Sarah; Nunez-Garcia, Javier; Steinbach, Falko; La Rocca, Anna; Blake, Damer; Dastjerdi, Akbar

    2014-10-01

    Pan viral DNA microarrays, which can detect known, novel and multiple viral infections, are major laboratory assets contributing to the control of infectious diseases. The large quantity of ribosomal RNA (rRNA) found in tissue samples is thought to be a major factor contributing to the comparatively lower sensitivity of detecting RNA viruses, as a sequence-independent PCR is used to amplify unknown samples for microarray analysis. This study aimed to determine whether depletion or exclusion of rRNA can improve microarray detection and simplify its analysis. Therefore, two different rRNA depletion and exclusion protocols, RiboMinus™ technology and non-rRNA binding hexanucleotides, were applied to the microarray sample processing and the outcome was compared with those of the sequence-independent amplification protocol. This study concludes that the two procedures, described to deplete or exclude rRNA, have negligible effect on the microarrays detection and analysis and might only in combination with further techniques result in a significant enhancement of sensitivity. Currently, existing protocols of random amplification and background adjustment are pertinent for the purpose of sample processing for microarray analysis.

  11. Viral diagnosis in Indian livestock using customized microarray chips.

    PubMed

    Yadav, Brijesh S; Pokhriyal, Mayank; Ratta, Barkha; Kumar, Ajay; Saxena, Meeta; Sharma, Bhaskar

    2015-01-01

    Viral diagnosis in Indian livestock using customized microarray chips is gaining momentum in recent years. Hence, it is possible to design customized microarray chip for viruses infecting livestock in India. Customized microarray chips identified Bovine herpes virus-1 (BHV-1), Canine Adeno Virus-1 (CAV-1), and Canine Parvo Virus-2 (CPV-2) in clinical samples. Microarray identified specific probes were further confirmed using RT-PCR in all clinical and known samples. Therefore, the application of microarray chips during viral disease outbreaks in Indian livestock is possible where conventional methods are unsuitable. It should be noted that customized application requires a detailed cost efficiency calculation.

  12. Advancing translational research with next-generation protein microarrays.

    PubMed

    Yu, Xiaobo; Petritis, Brianne; LaBaer, Joshua

    2016-04-01

    Protein microarrays are a high-throughput technology used increasingly in translational research, seeking to apply basic science findings to enhance human health. In addition to assessing protein levels, posttranslational modifications, and signaling pathways in patient samples, protein microarrays have aided in the identification of potential protein biomarkers of disease and infection. In this perspective, the different types of full-length protein microarrays that are used in translational research are reviewed. Specific studies employing these microarrays are presented to highlight their potential in finding solutions to real clinical problems. Finally, the criteria that should be considered when developing next-generation protein microarrays are provided. PMID:26749402

  13. Technical Advances of the Recombinant Antibody Microarray Technology Platform for Clinical Immunoproteomics

    PubMed Central

    Delfani, Payam; Dexlin Mellby, Linda; Nordström, Malin; Holmér, Andreas; Ohlsson, Mattias; Borrebaeck, Carl A. K.; Wingren, Christer

    2016-01-01

    In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by antibody-based microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant antibody microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant antibody microarray technology platform designed for clinical immunoproteomics. PMID:27414037

  14. Technical Advances of the Recombinant Antibody Microarray Technology Platform for Clinical Immunoproteomics.

    PubMed

    Delfani, Payam; Dexlin Mellby, Linda; Nordström, Malin; Holmér, Andreas; Ohlsson, Mattias; Borrebaeck, Carl A K; Wingren, Christer

    2016-01-01

    In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by antibody-based microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant antibody microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant antibody microarray technology platform designed for clinical immunoproteomics.

  15. PMD: A Resource for Archiving and Analyzing Protein Microarray data.

    PubMed

    Xu, Zhaowei; Huang, Likun; Zhang, Hainan; Li, Yang; Guo, Shujuan; Wang, Nan; Wang, Shi-Hua; Chen, Ziqing; Wang, Jingfang; Tao, Sheng-Ce

    2016-01-27

    Protein microarray is a powerful technology for both basic research and clinical study. However, because there is no database specifically tailored for protein microarray, the majority of the valuable original protein microarray data is still not publically accessible. To address this issue, we constructed Protein Microarray Database (PMD), which is specifically designed for archiving and analyzing protein microarray data. In PMD, users can easily browse and search the entire database by experimental name, protein microarray type, and sample information. Additionally, PMD integrates several data analysis tools and provides an automated data analysis pipeline for users. With just one click, users can obtain a comprehensive analysis report for their protein microarray data. The report includes preliminary data analysis, such as data normalization, candidate identification, and an in-depth bioinformatics analysis of the candidates, which include functional annotation, pathway analysis, and protein-protein interaction network analysis. PMD is now freely available at www.proteinmicroarray.cn.

  16. PMD: A Resource for Archiving and Analyzing Protein Microarray data

    PubMed Central

    Xu, Zhaowei; Huang, Likun; Zhang, Hainan; Li, Yang; Guo, Shujuan; Wang, Nan; Wang, Shi-hua; Chen, Ziqing; Wang, Jingfang; Tao, Sheng-ce

    2016-01-01

    Protein microarray is a powerful technology for both basic research and clinical study. However, because there is no database specifically tailored for protein microarray, the majority of the valuable original protein microarray data is still not publically accessible. To address this issue, we constructed Protein Microarray Database (PMD), which is specifically designed for archiving and analyzing protein microarray data. In PMD, users can easily browse and search the entire database by experimental name, protein microarray type, and sample information. Additionally, PMD integrates several data analysis tools and provides an automated data analysis pipeline for users. With just one click, users can obtain a comprehensive analysis report for their protein microarray data. The report includes preliminary data analysis, such as data normalization, candidate identification, and an in-depth bioinformatics analysis of the candidates, which include functional annotation, pathway analysis, and protein-protein interaction network analysis. PMD is now freely available at www.proteinmicroarray.cn. PMID:26813635

  17. 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

  18. Statistical Considerations for Analysis of Microarray Experiments

    PubMed Central

    Owzar, Kouros; Barry, William T.; Jung, Sin-Ho

    2014-01-01

    Microarray technologies enable the simultaneous interrogation of expressions from thousands of genes from a biospecimen sample taken from a patient. This large set of expressions generate a genetic profile of the patient that may be used to identify potential prognostic or predictive genes or genetic models for clinical outcomes. The aim of this article is to provide a broad overview of some of the major statistical considerations for the design and analysis of microarrays experiments conducted as correlative science studies to clinical trials. An emphasis will be placed on how the lack of understanding and improper use of statistical concepts and methods will lead to noise discovery and misinterpretation of experimental results. PMID:22212230

  19. Plasmonically amplified fluorescence bioassay with microarray format

    NASA Astrophysics Data System (ADS)

    Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.

    2015-05-01

    Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

  20. Microarrays: how many do you need?

    PubMed

    Zien, Alexander; Fluck, Juliane; Zimmer, Ralf; Lengauer, Thomas

    2003-01-01

    We estimate the number of microarrays that is required in order to gain reliable results from a common type of study: the pairwise comparison of different classes of samples. We show that current knowledge allows for the construction of models that look realistic with respect to searches for individual differentially expressed genes and derive prototypical parameters from real data sets. Such models allow investigation of the dependence of the required number of samples on the relevant parameters: the biological variability of the samples within each class, the fold changes in expression that are desired to be detected, the detection sensitivity of the microarrays, and the acceptable error rates of the results. We supply experimentalists with general conclusions as well as a freely accessible Java applet at www.scai.fhg.de/special/bio/howmanyarrays/ for fine tuning simulations to their particular settings. PMID:12935350

  1. A Flexible Microarray Data Simulation Model

    PubMed Central

    Dembélé, Doulaye

    2013-01-01

    Microarray technology allows monitoring of gene expression profiling at the genome level. This is useful in order to search for genes involved in a disease. The performances of the methods used to select interesting genes are most often judged after other analyzes (qPCR validation, search in databases...), which are also subject to error. A good evaluation of gene selection methods is possible with data whose characteristics are known, that is to say, synthetic data. We propose a model to simulate microarray data with similar characteristics to the data commonly produced by current platforms. The parameters used in this model are described to allow the user to generate data with varying characteristics. In order to show the flexibility of the proposed model, a commented example is given and illustrated. An R package is available for immediate use.

  2. Profiling protein function with small molecule microarrays

    PubMed Central

    Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

    2002-01-01

    The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

  3. Application of DNA Microarray to Clinical Diagnostics.

    PubMed

    Patel, Ankita; Cheung, Sau W

    2016-01-01

    Microarray-based technology to conduct array comparative genomic hybridization (aCGH) has made a significant impact on the diagnosis of human genetic diseases. Such diagnoses, previously undetectable by traditional G-banding chromosome analysis, are now achieved by identifying genomic copy number variants (CNVs) using the microarray. Not only can hundreds of well-characterized genetic syndromes be detected in a single assay, but new genomic disorders and disease-causing genes can also be discovered through the utilization of aCGH technology. Although other platforms such as single nucleotide polymorphism (SNP) arrays can be used for detecting CNVs, in this chapter we focus on describing the methods for performing aCGH using Agilent oligonucleotide arrays for both prenatal (e.g., amniotic fluid and chorionic villus sample) and postnatal samples (e.g., blood).

  4. Weighted analysis of general microarray experiments

    PubMed Central

    Sjögren, Anders; Kristiansson, Erik; Rudemo, Mats; Nerman, Olle

    2007-01-01

    Background In DNA microarray experiments, measurements from different biological samples are often assumed to be independent and to have identical variance. For many datasets these assumptions have been shown to be invalid and typically lead to too optimistic p-values. A method called WAME has been proposed where a variance is estimated for each sample and a covariance is estimated for each pair of samples. The current version of WAME is, however, limited to experiments with paired design, e.g. two-channel microarrays. Results The WAME procedure is extended to general microarray experiments, making it capable of handling both one- and two-channel datasets. Two public one-channel datasets are analysed and WAME detects both unequal variances and correlations. WAME is compared to other common methods: fold-change ranking, ordinary linear model with t-tests, LIMMA and weighted LIMMA. The p-value distributions are shown to differ greatly between the examined methods. In a resampling-based simulation study, the p-values generated by WAME are found to be substantially more correct than the alternatives when a relatively small proportion of the genes is regulated. WAME is also shown to have higher power than the other methods. WAME is available as an R-package. Conclusion The WAME procedure is generalized and the limitation to paired-design microarray datasets is removed. The examined other methods produce invalid p-values in many cases, while WAME is shown to produce essentially valid p-values when a relatively small proportion of genes is regulated. WAME is also shown to have higher power than the examined alternative methods. PMID:17937807

  5. Undetected sex chromosome aneuploidy by chromosomal microarray.

    PubMed

    Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

    2012-11-01

    We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting.

  6. VAMPIRE microarray suite: a web-based platform for the interpretation of gene expression data.

    PubMed

    Hsiao, Albert; Ideker, Trey; Olefsky, Jerrold M; Subramaniam, Shankar

    2005-07-01

    Microarrays are invaluable high-throughput tools used to snapshot the gene expression profiles of cells and tissues. Among the most basic and fundamental questions asked of microarray data is whether individual genes are significantly activated or repressed by a particular stimulus. We have previously presented two Bayesian statistical methods for this level of analysis, collectively known as variance-modeled posterior inference with regional exponentials (VAMPIRE). These methods each require a sophisticated modeling step followed by integration of a posterior probability density. We present here a publicly available, web-based platform that allows users to easily load data, associate related samples and identify differentially expressed features using the VAMPIRE statistical framework. In addition, this suite of tools seamlessly integrates a novel gene annotation tool, known as GOby, which identifies statistically overrepresented gene groups. Unlike other tools in this genre, GOby can localize enrichment while respecting the hierarchical structure of annotation systems like Gene Ontology (GO). By identifying statistically significant enrichment of GO terms, Kyoto Encyclopedia of Genes and Genomes pathways, and TRANSFAC transcription factor binding sites, users can gain substantial insight into the physiological significance of sets of differentially expressed genes. The VAMPIRE microarray suite can be accessed at http://genome.ucsd.edu/microarray.

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

    PubMed Central

    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. PMID:27082962

  8. Analysis of discordant Affymetrix probesets casts serious doubt on idea of microarray data reutilization

    PubMed Central

    2014-01-01

    Background Affymetrix microarray technology allows one to investigate expression of thousands of genes simultaneously upon a variety of conditions. In a popular U133A microarray platform, the expression of 37% of genes is measured by more than one probeset. The discordant expression observed for two different probesets that match the same gene is a widespread phenomenon which is usually underestimated, ignored or disregarded. Results Here we evaluate the prevalence of discordant expression in data collected using Affymetrix HG-U133A microarray platform. In U133A, about 30% of genes annotated by two different probesets demonstrate a substantial correlation between independently measured expression values. To our surprise, sorting the probesets according to the nature of the discrepancy in their expression levels allowed the classification of the respective genes according to their fundamental functional properties, including observed enrichment by tissue-specific transcripts and alternatively spliced variants. On another hand, an absence of discrepancies in probesets that simultaneously match several different genes allowed us to pinpoint non-expressed pseudogenes and gene groups with highly correlated expression patterns. Nevertheless, in many cases, the nature of discordant expression of two probesets that match the same transcript remains unexplained. It is possible that these probesets report differently regulated sets of transcripts, or, in best case scenario, two different sets of transcripts that represent the same gene. Conclusion The majority of absolute gene expression values collected using Affymetrix microarrays may not be suitable for typical interpretative downstream analysis. PMID:25563078

  9. 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.

  10. Microarray analysis in gastric cancer: A review

    PubMed Central

    D’Angelo, Giovanna; Di Rienzo, Teresa; Ojetti, Veronica

    2014-01-01

    Gastric cancer is one of the most common tumors worldwide. Although several treatment options have been developed, the mortality rate is increasing. Lymph node involvement is considered the most reliable prognostic indicator in gastric cancer. Early diagnosis improves the survival rate of patients and increases the likelihood of successful treatment. The most reliable diagnostic method is endoscopic examination, however, it is expensive and not feasible in poorer countries. Therefore, many innovative techniques have been studied to develop a new non-invasive screening test and to identify specific serum biomarkers. DNA microarray analysis is one of the new technologies able to measure the expression levels of a large number of genes simultaneously. It is possible to define the gene expression profile of the tumor and to correlate it with the prognosis and metastasis formation. Several studies in the literature have been published on the role of microarray analysis in gastric cancer and the mechanisms of proliferation and metastasis formation. The aim of this review is to analyze the importance of microarray analysis and its clinical applications to better define the genetic characteristics of gastric cancer and its possible implications in a more decisive treatment. PMID:25232233

  11. Repeatability of published microarray gene expression analyses.

    PubMed

    Ioannidis, John P A; Allison, David B; Ball, Catherine A; Coulibaly, Issa; Cui, Xiangqin; Culhane, Aedín C; Falchi, Mario; Furlanello, Cesare; Game, Laurence; Jurman, Giuseppe; Mangion, Jon; Mehta, Tapan; Nitzberg, Michael; Page, Grier P; Petretto, Enrico; van Noort, Vera

    2009-02-01

    Given the complexity of microarray-based gene expression studies, guidelines encourage transparent design and public data availability. Several journals require public data deposition and several public databases exist. However, not all data are publicly available, and even when available, it is unknown whether the published results are reproducible by independent scientists. Here we evaluated the replication of data analyses in 18 articles on microarray-based gene expression profiling published in Nature Genetics in 2005-2006. One table or figure from each article was independently evaluated by two teams of analysts. We reproduced two analyses in principle and six partially or with some discrepancies; ten could not be reproduced. The main reason for failure to reproduce was data unavailability, and discrepancies were mostly due to incomplete data annotation or specification of data processing and analysis. Repeatability of published microarray studies is apparently limited. More strict publication rules enforcing public data availability and explicit description of data processing and analysis should be considered.

  12. An imputation approach for oligonucleotide microarrays.

    PubMed

    Li, Ming; Wen, Yalu; Lu, Qing; Fu, Wenjiang J

    2013-01-01

    Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as "bright spots", "dark clouds", and "shadowy circles", etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.

  13. High-Throughput Enzyme Kinetics Using Microarrays

    SciTech Connect

    Guoxin Lu; Edward S. Yeung

    2007-11-01

    We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)- coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant K{sub m} obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner.

  14. Integrating data from heterogeneous DNA microarray platforms.

    PubMed

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus. PMID:26673932

  15. Development and Applications of the Lectin Microarray.

    PubMed

    Hirabayashi, Jun; Kuno, Atsushi; Tateno, Hiroaki

    2015-01-01

    The lectin microarray is an emerging technology for glycomics. It has already found maximum use in diverse fields of glycobiology by providing simple procedures for differential glycan profiling in a rapid and high-throughput manner. Since its first appearance in the literature in 2005, many application methods have been developed essentially on the same platform, comprising a series of glycan-binding proteins immobilized on an appropriate substrate such as a glass slide. Because the lectin microarray strategy does not require prior liberation of glycans from the core protein in glycoprotein analysis, it should encourage researchers not familiar with glycotechnology to use glycan analysis in future work. This feasibility should provide a broader range of experimental scientists with good opportunities to investigate novel aspects of glycoscience. Applications of the technology include not only basic sciences but also the growing fields of bio-industry. This chapter describes first the essence of glycan profiling and the basic fabrication of the lectin microarray for this purpose. In the latter part the focus is on diverse applications to both structural and functional glycomics, with emphasis on the wide applicability now available with this new technology. Finally, the importance of developing advanced lectin engineering is discussed.

  16. 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. PMID:24052712

  17. Chicken sperm transcriptome profiling by microarray analysis.

    PubMed

    Singh, R P; Shafeeque, C M; Sharma, S K; Singh, R; Mohan, J; Sastry, K V H; Saxena, V K; Azeez, P A

    2016-03-01

    It has been confirmed that mammalian sperm contain thousands of functional RNAs, and some of them have vital roles in fertilization and early embryonic development. Therefore, we attempted to characterize transcriptome of the sperm of fertile chickens using microarray analysis. Spermatozoal RNA was pooled from 10 fertile males and used for RNA preparation. Prior to performing the microarray, RNA quality was assessed using a bioanalyzer, and gDNA and somatic cell RNA contamination was assessed by CD4 and PTPRC gene amplification. The chicken sperm transcriptome was cross-examined by analysing sperm and testes RNA on a 4 × 44K chicken array, and results were verified by RT-PCR. Microarray analysis identified 21,639 predominantly nuclear-encoded transcripts in chicken sperm. The majority (66.55%) of the sperm transcripts were shared with the testes, while surprisingly, 33.45% transcripts were detected (raw signal intensity greater than 50) only in the sperm and not in the testes. The greatest proportion of up-regulated transcripts were responsible for signal transduction (63.20%) followed by embryonic development (56.76%) and cell structure (56.25%). Of the 20 most abundant transcripts, 18 remain uncharacterized, whereas the least abundant genes were mostly associated with the ribosome. These findings lay a foundation for more detailed investigations on sperm RNAs in chickens to identify sperm-based biomarkers for fertility.

  18. [Genomic medicine. Polymorphisms and microarray applications].

    PubMed

    Spalvieri, Mónica P; Rotenberg, Rosa G

    2004-01-01

    This update shows new concepts related to the significance of DNA variations among individuals, as well as to their detection by using a new technology. The sequencing of the human genome is only the beginning of what will enable us to understand genetic diversity. The unit of DNA variability is the polymorphism of a single nucleotide (SNP). At present, studies on SNPs are restricted to basic research but the large number of papers on this subject makes feasible their entrance into clinical practice. We illustrate here the use of SNPs as molecular markers in ethnical genotyping, gene expression in some diseases and as potential targets in pharmacological response, and also introduce the technology of arrays. Microarrays experiments allow the quantification and comparison of gene expression on a large scale, at the same time, by using special chips and array designs. Conventional methods provide data from up to 20 genes, while a single microarray may provide information about thousands of them simultaneously, leading to a more rapid and accurate genotyping. Biotechnology improvements will facilitate our knowledge of each gene sequence, the frequency and exact location of SNPs and their influence on cellular behavior. Although experimental efficiency and validity of results from microarrays are still controversial, the knowledge and characterization of a patient's genetic profile will lead, undoubtedly, to advances in prevention, diagnosis, prognosis and treatment of human diseases. PMID:15637833

  19. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  20. DNA microarray for detection of gastrointestinal viruses.

    PubMed

    Martínez, Miguel A; Soto-Del Río, María de Los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y; Greninger, Alexander L; Contreras, Juan Francisco; López, Susana; Arias, Carlos F; Isa, Pavel

    2015-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  1. Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes.

    PubMed

    Akimoto, Yoshihiro; Takata, Kuniaki; Kawakami, Hayato

    2016-01-01

    In recent years, the study of glycans is progressing remarkably by the development of glycan analysis systems using mass spectrometry, glycan profiling systems using lectin microarrays, and glycoprotein analysis by the isotope-coded glycosylation site-specific tagging method. With these methodologies, glycan structures and biological functions are being elucidated. In the study of glycan function as well as disease diagnosis, it is important to examine the localization of glycans in tissues and cells. Histochemical methods using lectin probes can localize glycans in the tissues and cells. This chapter describes a pre-embedding electron microscopic method for glycan localization in which tissue sections and cells are incubated with lectin prior to embedding in resin. PMID:27515086

  2. 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.

  3. Statistically designing microarrays and microarray experiments to enhance sensitivity and specificity.

    PubMed

    Hsu, Jason C; Chang, Jane; Wang, Tao; Steingrímsson, Eiríkur; Magnússon, Magnús Karl; Bergsteinsdottir, Kristin

    2007-01-01

    Gene expression signatures from microarray experiments promise to provide important prognostic tools for predicting disease outcome or response to treatment. A number of microarray studies in various cancers have reported such gene signatures. However, the overlap of gene signatures in the same disease has been limited so far, and some reported signatures have not been reproduced in other populations. Clearly, the methods used for verifying novel gene signatures need improvement. In this article, we describe an experiment in which microarrays and sample hybridization are designed according to the statistical principles of randomization, replication and blocking. Our results show that such designs provide unbiased estimation of differential expression levels as well as powerful tests for them.

  4. Comments on selected fundamental aspects of microarray analysis.

    PubMed

    Riva, Alessandra; Carpentier, Anne-Sophie; Torrésani, Bruno; Hénaut, Alain

    2005-10-01

    Microarrays are becoming a ubiquitous tool of research in life sciences. However, the working principles of microarray-based methodologies are often misunderstood or apparently ignored by the researchers who actually perform and interpret experiments. This in turn seems to lead to a common over-expectation regarding the explanatory and/or knowledge-generating power of microarray analyses. In this note we intend to explain basic principles of five (5) major groups of analytical techniques used in studies of microarray data and their interpretation: the principal component analysis (PCA), the independent component analysis (ICA), the t-test, the analysis of variance (ANOVA), and self organizing maps (SOM). We discuss answers to selected practical questions related to the analysis of microarray data. We also take a closer look at the experimental setup and the rules, which have to be observed in order to exploit microarrays efficiently. Finally, we discuss in detail the scope and limitations of microarray-based methods. We emphasize the fact that no amount of statistical analysis can compensate for (or replace) a well thought through experimental setup. We conclude that microarrays are indeed useful tools in life sciences but by no means should they be expected to generate complete answers to complex biological questions. We argue that even well posed questions, formulated within a microarray-specific terminology, cannot be completely answered with the use of microarray analyses alone.

  5. Microarray analysis of gene expression in mouse (strain 129) embryonic stem cells after typical synthetic musk exposure.

    PubMed

    Shi, Jiachen; Li, Ming; Jiao, Zhihao; Zhang, Jing; Feng, Yixing; Shao, Bing

    2013-01-01

    Synthetic musks are widely used in personal-care products and can readily accumulate in the adipose tissue, breast milk, and blood of humans. In this study, the Affymetrix Mouse Genome GeneChip was used to identify alterations in gene expression of embryonic stem cells from the 129 strain of the laboratory mouse after treatment with the synthetic musk tonalide (AHTN). Among the 45,037 transcripts in the microarray, 2,879 genes were differentially expressed. According to the microarray analysis, the potential influence of AHTN on the development to embryo should be of concern, and the toxicological effects of it and related musk compounds should be studied further.

  6. An algorithm for finding biologically significant features in microarray data based on a priori manifold learning.

    PubMed

    Hira, Zena M; Trigeorgis, George; Gillies, Duncan F

    2014-01-01

    Microarray databases are a large source of genetic data, which, upon proper analysis, could enhance our understanding of biology and medicine. Many microarray experiments have been designed to investigate the genetic mechanisms of cancer, and analytical approaches have been applied in order to classify different types of cancer or distinguish between cancerous and non-cancerous tissue. However, microarrays are high-dimensional datasets with high levels of noise and this causes problems when using machine learning methods. A popular approach to this problem is to search for a set of features that will simplify the structure and to some degree remove the noise from the data. The most widely used approach to feature extraction is principal component analysis (PCA) which assumes a multivariate Gaussian model of the data. More recently, non-linear methods have been investigated. Among these, manifold learning algorithms, for example Isomap, aim to project the data from a higher dimensional space onto a lower dimension one. We have proposed a priori manifold learning for finding a manifold in which a representative set of microarray data is fused with relevant data taken from the KEGG pathway database. Once the manifold has been constructed the raw microarray data is projected onto it and clustering and classification can take place. In contrast to earlier fusion based methods, the prior knowledge from the KEGG databases is not used in, and does not bias the classification process--it merely acts as an aid to find the best space in which to search the data. In our experiments we have found that using our new manifold method gives better classification results than using either PCA or conventional Isomap. PMID:24595155

  7. 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. PMID:15574946

  8. 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

  9. Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model.

    PubMed

    Cabalén, María E; Cabral, María F; Sanmarco, Liliana M; Andrada, Marta C; Onofrio, Luisina I; Ponce, Nicolás E; Aoki, María P; Gea, Susana; Cano, Roxana C

    2016-03-22

    Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4-/- mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression. PMID:26921251

  10. Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model

    PubMed Central

    Cabalén, María E.; Cabral, María F.; Sanmarco, Liliana M.; Andrada, Marta C.; Onofrio, Luisina I.; Ponce, Nicolás E.; Aoki, María P.; Gea, Susana; Cano, Roxana C.

    2016-01-01

    Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4−/− mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression. PMID:26921251

  11. Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model.

    PubMed

    Cabalén, María E; Cabral, María F; Sanmarco, Liliana M; Andrada, Marta C; Onofrio, Luisina I; Ponce, Nicolás E; Aoki, María P; Gea, Susana; Cano, Roxana C

    2016-03-22

    Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4-/- mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression.

  12. Rapid characterization of candidate biomarkers for pancreatic cancer using cell microarrays (CMAs).

    PubMed

    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

    2012-11-01

    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.

  13. High-throughput allogeneic antibody detection using protein microarrays.

    PubMed

    Paul, Jed; Sahaf, Bita; Perloff, Spenser; Schoenrock, Kelsi; Wu, Fang; Nakasone, Hideki; Coller, John; Miklos, David

    2016-05-01

    Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform. PMID:26902899

  14. Formation and characterization of DNA microarrays at silicon nitride substrates.

    PubMed

    Manning, Mary; Redmond, Gareth

    2005-01-01

    A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.

  15. Tissues from the irradiated dog/mouse archive

    SciTech Connect

    Gayle Woloschak

    2007-04-01

    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 inflammatory 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

  16. A longitudinal study of MARS MRI scanning of soft-tissue lesions around metal-on-metal total hip arthroplasties and disease progression.

    PubMed

    Briant-Evans, T W; Lyle, N; Barbur, S; Hauptfleisch, J; Amess, R; Pearce, A R; Conn, K S; Stranks, G J; Britton, J M

    2015-10-01

    We investigated the changes seen on serial metal artefact reduction magnetic resonance imaging scans (MARS-MRI) of metal-on-metal total hip arthroplasties (MoM THAs). In total 155 THAs, in 35 male and 100 female patients (mean age 70.4 years, 42 to 91), underwent at least two MRI scans at a mean interval of 14.6 months (2.6 to 57.1), at a mean of 48.2 months (3.5 to 93.3) after primary hip surgery. Scans were graded using a modification of the Oxford classification. Progression of disease was defined as an increase in grade or a minimum 10% increase in fluid lesion volume at second scan. A total of 16 hips (30%) initially classified as 'normal' developed an abnormality on the second scan. Of those with 'isolated trochanteric fluid' 9 (47%) underwent disease progression, as did 7 (58%) of 'effusions'. A total of 54 (77%) of hips initially classified as showing adverse reactions to metal debris (ARMD) progressed, with higher rates of progression in higher grades. Disease progression was associated with high blood cobalt levels or an irregular pseudocapsule lining at the initial scan. There was no association with changes in functional scores. Adverse reactions to metal debris in MoM THAs may not be as benign as previous reports have suggested. Close radiological follow-up is recommended, particularly in high-risk groups. PMID:26430006

  17. A longitudinal study of MARS MRI scanning of soft-tissue lesions around metal-on-metal total hip arthroplasties and disease progression.

    PubMed

    Briant-Evans, T W; Lyle, N; Barbur, S; Hauptfleisch, J; Amess, R; Pearce, A R; Conn, K S; Stranks, G J; Britton, J M

    2015-10-01

    We investigated the changes seen on serial metal artefact reduction magnetic resonance imaging scans (MARS-MRI) of metal-on-metal total hip arthroplasties (MoM THAs). In total 155 THAs, in 35 male and 100 female patients (mean age 70.4 years, 42 to 91), underwent at least two MRI scans at a mean interval of 14.6 months (2.6 to 57.1), at a mean of 48.2 months (3.5 to 93.3) after primary hip surgery. Scans were graded using a modification of the Oxford classification. Progression of disease was defined as an increase in grade or a minimum 10% increase in fluid lesion volume at second scan. A total of 16 hips (30%) initially classified as 'normal' developed an abnormality on the second scan. Of those with 'isolated trochanteric fluid' 9 (47%) underwent disease progression, as did 7 (58%) of 'effusions'. A total of 54 (77%) of hips initially classified as showing adverse reactions to metal debris (ARMD) progressed, with higher rates of progression in higher grades. Disease progression was associated with high blood cobalt levels or an irregular pseudocapsule lining at the initial scan. There was no association with changes in functional scores. Adverse reactions to metal debris in MoM THAs may not be as benign as previous reports have suggested. Close radiological follow-up is recommended, particularly in high-risk groups.

  18. RNA-seq as a powerful tool for penaeid shrimp genetic progress

    PubMed Central

    Santos, Camilla A.; Blanck, Danielly V.; de Freitas, Patrícia D.

    2014-01-01

    The sequences of all different RNA transcripts present in a cell or tissue that are related to the gene expression and its functional control represent what it is called a transcriptome. The transcripts vary between cells, tissues, ontogenetic and environmental conditions, and the knowledge that can be gained through them is of a solid relevance for genetic applications in aquaculture. Some of the techniques used in transcriptome studies, such as microarrays, are being replaced for next-generation sequencing approaches. RNA-seq emerges as a new possibility for the transcriptome complexity analysis as well as for the candidate genes and polymorphisms identification of penaeid species. Thus, it may also help to understand the determination of complex traits mechanisms and genetic improvement of stocks. In this review, it is first introduced an overview of transcriptome analysis by RNA-seq, followed by a discussion of how this approach may be applied in genetic progress within penaeid stocks. PMID:25221571

  19. Genetics Home Reference: progressive osseous heteroplasia

    MedlinePlus

    ... and muscle tissue. Bone that forms outside the skeleton is called heterotopic or ectopic bone. In progressive ... preventing bony tissue from being produced outside the skeleton. The GNAS gene mutations that cause progressive osseous ...

  20. ProMAT: protein microarray analysis tool

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Varnum, Susan M.; Anderson, Kevin K.; Bollinger, Nikki; Zangar, Richard C.

    2006-04-04

    Summary: ProMAT is a software tool for statistically analyzing data from ELISA microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality. The tool is available for Windows or Mac, and is distributed as open-source Java and R code. Availability: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions) which are distributed with the tool.

  1. Protein Microarrays--Without a Trace

    SciTech Connect

    Camarero, J A

    2007-04-05

    Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.

  2. Identification of plant defence genes in canola using Arabidopsis cDNA microarrays.

    PubMed

    Schenk, P M; Thomas-Hall, S R; Nguyen, A V; Manners, J M; Kazan, K; Spangenberg, G

    2008-09-01

    We report the identification of novel defence genes in canola by using a cDNA microarray from Arabidopsis. We examined changes that occur in the abundance of transcripts corresponding to 2375 Arabidopsis expressed sequence tags (selected for defence gene identification) following inoculation of canola plants with the fungal necrotrophic leaf pathogen, Alternaria brassicicola. Microarray data obtained from this cross-hybridisation experiment were compared to expression profiles previously obtained from the equivalent Arabidopsis experiment. Homology searches using a canola expressed sequence tag database with approximately 6000 unique clones led to identification of canola defence genes. Pathogen-responsive transcripts included those associated to known defence genes, reactive oxygen species metabolism, disease resistance and regulatory genes, and cell maintenance/metabolism genes. Using specific primers for quantitative real-time reverse transcriptase PCR, gene expression profiles in canola were obtained that demonstrated coordinated defence responses, including systemic responses in distal tissue and salicylic acid- and methyl jasmonate-mediated signalling against A. brassicicola.

  3. A custom microarray platform for analysis of microRNA gene expression.

    PubMed

    Thomson, J Michael; Parker, Joel; Perou, Charles M; Hammond, Scott M

    2004-10-01

    MicroRNAs are short, noncoding RNA transcripts that post-transcriptionally regulate gene expression. Several hundred microRNA genes have been identified in Caenorhabditis elegans, Drosophila, plants and mammals. MicroRNAs have been linked to developmental processes in C. elegans, plants and humans and to cell growth and apoptosis in Drosophila. A major impediment in the study of microRNA function is the lack of quantitative expression profiling methods. To close this technological gap, we have designed dual-channel microarrays that monitor expression levels of 124 mammalian microRNAs. Using these tools, we observed distinct patterns of expression among adult mouse tissues and embryonic stem cells. Expression profiles of staged embryos demonstrate temporal regulation of a large class of microRNAs, including members of the let-7 family. This microarray technology enables comprehensive investigation of microRNA expression, and furthers our understanding of this class of recently discovered noncoding RNAs.

  4. The role of APE/Ref-1 signaling pathway in hepatocellular carcinoma progression.

    PubMed

    Yang, Zhen; Yang, Sun; Misner, Bobbye J; Liu-Smith, Feng; Meyskens, Frank L

    2014-11-01

    Hepatocellular carcinoma (HCC) is responsible for a third of the estimated cancer-caused deaths worldwide. To deeply understand the mechanisms controlling HCC progression is of primary importance to develop new approaches for treatment. Apurinic/apyrimidinic endonuclease-1/redox effector factor 1 (APE/Ref-1) has been uncovered elevated in various types of cancer, including HCC. Additionally, HCC progression is always correlated with elevated copper (Cu). Our previous data demonstrated that Cu treatment initiated APE/Ref-1 expression and its downstream targets. Therefore, we hypothesized that APE/Ref-1 may be involved in HCC progression through mediating the effect of Cu to its signaling cascades. Following different treatments, human HCC cell line (Hep3B) and immortalized non-malignant hepatocyte cell line (THLE3) were analyzed to explore the role of APE/Ref-1 signaling pathway. Unstained human tissue microarrays (TMA) were subjected to IHC analysis to study the relationship between APE/Ref-1 expression and clinic features. APE/Ref-1 was upregulated in HCC cells consistent with the strong expression of APE/Ref-1 in HCC tissue microarray. Greater cytoplasmic accumulation of APE/Ref-1 was found in poorly differentiated and more aggressive tumors. Also we provide evidence to show that APE/Ref-1 signaling pathway stimulates cellular proliferation, enhances anti-apoptosis, and facilitates metastasis through experimental knockdown of APE/Ref-1 using siRNA in Hep3B cells or overexpressing APE/Ref-1 in THLE3 cells. These results define a novel role of APE/Ref-1 in HCC progression as being an important mediating and potentiating molecule, and also provide a basis for further investigations utilizing appropriate APE/Ref-1 inhibitors in combination with chemo-drugs for HCC treatment.

  5. Refractive index change detection based on porous silicon microarray

    NASA Astrophysics Data System (ADS)

    Chen, Weirong; Jia, Zhenhong; Li, Peng; Lv, Guodong; Lv, Xiaoyi

    2016-05-01

    By combining photolithography with the electrochemical anodization method, a microarray device of porous silicon (PS) photonic crystal was fabricated on the crystalline silicon substrate. The optical properties of the microarray were analyzed with the transfer matrix method. The relationship between refractive index and reflectivity of each array element of the microarray at 633 nm was also studied, and the array surface reflectivity changes were observed through digital imaging. By means of the reflectivity measurement method, reflectivity changes below 10-3 can be observed based on PS microarray. The results of this study can be applied to the detection of biosensor arrays.

  6. Re-Annotator: Annotation Pipeline for Microarray Probe Sequences.

    PubMed

    Arloth, Janine; Bader, Daniel M; Röh, Simone; Altmann, Andre

    2015-01-01

    Microarray technologies are established approaches for high throughput gene expression, methylation and genotyping analysis. An accurate mapping of the array probes is essential to generate reliable biological findings. However, manufacturers of the microarray platforms typically provide incomplete and outdated annotation tables, which often rely on older genome and transcriptome versions that differ substantially from up-to-date sequence databases. Here, we present the Re-Annotator, a re-annotation pipeline for microarray probe sequences. It is primarily designed for gene expression microarrays but can also be adapted to other types of microarrays. The Re-Annotator uses a custom-built mRNA reference database to identify the positions of gene expression array probe sequences. We applied Re-Annotator to the Illumina Human-HT12 v4 microarray platform and found that about one quarter (25%) of the probes differed from the manufacturer's annotation. In further computational experiments on experimental gene expression data, we compared Re-Annotator to another probe re-annotation tool, ReMOAT, and found that Re-Annotator provided an improved re-annotation of microarray probes. A thorough re-annotation of probe information is crucial to any microarray analysis. The Re-Annotator pipeline is freely available at http://sourceforge.net/projects/reannotator along with re-annotated files for Illumina microarrays HumanHT-12 v3/v4 and MouseRef-8 v2.

  7. Chemiluminescence microarrays in analytical chemistry: a critical review.

    PubMed

    Seidel, Michael; Niessner, Reinhard

    2014-09-01

    Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

  8. Studying cellular processes and detecting disease with protein microarrays

    SciTech Connect

    Zangar, Richard C.; Varnum, Susan M.; Bollinger, Nikki

    2005-10-31

    Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take. In this review, we provide a short, conceptual overview of the different approaches for protein microarray. We then examine some of the most significant applications of these microarrays to date, with an emphasis on how global protein analyses can be used to facilitate biomedical research.

  9. The use of antigen microarrays in antibody profiling.

    PubMed

    Papp, Krisztián; Prechl, József

    2012-01-01

    Technological advances in the field of microarray production and analysis lead to the development of protein microarrays. Of these, antigen microarrays are one particular format that allows the study of antigen-antibody interactions in a miniaturized and highly multiplexed fashion. Here, we describe the parallel detection of antibodies with different specificities in human serum, a procedure also called antibody profiling. Autoantigens printed on microarray slides are reacted with test sera and the bound antibodies are identified by fluorescently labeled secondary reagents. Reactivity patterns generated this way characterize individuals and can help design novel diagnostic tools.

  10. Sample phenotype clusters in high-density oligonucleotide microarray data sets are revealed using Isomap, a nonlinear algorithm

    PubMed Central

    Dawson, Kevin; Rodriguez, Raymond L; Malyj, Wasyl

    2005-01-01

    Background Life processes are determined by the organism's genetic profile and multiple environmental variables. However the interaction between these factors is inherently non-linear [1]. Microarray data is one representation of the nonlinear interactions among genes and genes and environmental factors. Still most microarray studies use linear methods for the interpretation of nonlinear data. In this study, we apply Isomap, a nonlinear method of dimensionality reduction, to analyze three independent large Affymetrix high-density oligonucleotide microarray data sets. Results Isomap discovered low-dimensional structures embedded in the Affymetrix microarray data sets. These structures correspond to and help to interpret biological phenomena present in the data. This analysis provides examples of temporal, spatial, and functional processes revealed by the Isomap algorithm. In a spinal cord injury data set, Isomap discovers the three main modalities of the experiment – location and severity of the injury and the time elapsed after the injury. In a multiple tissue data set, Isomap discovers a low-dimensional structure that corresponds to anatomical locations of the source tissues. This model is capable of describing low- and high-resolution differences in the same model, such as kidney-vs.-brain and differences between the nuclei of the amygdala, respectively. In a high-throughput drug screening data set, Isomap discovers the monocytic and granulocytic differentiation of myeloid cells and maps several chemical compounds on the two-dimensional model. Conclusion Visualization of Isomap models provides useful tools for exploratory analysis of microarray data sets. In most instances, Isomap models explain more of the variance present in the microarray data than PCA or MDS. Finally, Isomap is a promising new algorithm for class discovery and class prediction in high-density oligonucleotide data sets. PMID:16076401

  11. 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

  12. Inferring genetic networks from microarray data.

    SciTech Connect

    May, Elebeoba Eni; Davidson, George S.; Martin, Shawn Bryan; Werner-Washburne, Margaret C.; Faulon, Jean-Loup Michel

    2004-06-01

    In theory, it should be possible to infer realistic genetic networks from time series microarray data. In practice, however, network discovery has proved problematic. The three major challenges are: (1) inferring the network; (2) estimating the stability of the inferred network; and (3) making the network visually accessible to the user. Here we describe a method, tested on publicly available time series microarray data, which addresses these concerns. The inference of genetic networks from genome-wide experimental data is an important biological problem which has received much attention. Approaches to this problem have typically included application of clustering algorithms [6]; the use of Boolean networks [12, 1, 10]; the use of Bayesian networks [8, 11]; and the use of continuous models [21, 14, 19]. Overviews of the problem and general approaches to network inference can be found in [4, 3]. Our approach to network inference is similar to earlier methods in that we use both clustering and Boolean network inference. However, we have attempted to extend the process to better serve the end-user, the biologist. In particular, we have incorporated a system to assess the reliability of our network, and we have developed tools which allow interactive visualization of the proposed network.

  13. Intensity-based segmentation of microarray images.

    PubMed

    Nagarajan, Radhakrishnan

    2003-07-01

    The underlying principle in microarray image analysis is that the spot intensity is a measure of the gene expression. This implicitly assumes the gene expression of a spot to be governed entirely by the distribution of the pixel intensities. Thus, a segmentation technique based on the distribution of the pixel intensities is appropriate for the current problem. In this paper, clustering-based segmentation is described to extract the target intensity of the spots. The approximate boundaries of the spots in the microarray are determined by manual adjustment of rectilinear grids. The distribution of the pixel intensity in a grid containing a spot is assumed to be the superposition of the foreground and the local background. The k-means clustering technique and the partitioning around medoids (PAM) were used to generate a binary partition of the pixel intensity distribution. The median (k-means) and the medoid (PAM) of the cluster members are chosen as the cluster representatives. The effectiveness of the clustering-based segmentation techniques was tested on publicly available arrays generated in a lipid metabolism experiment (Callow et al., 2000). The results are compared against those obtained using the region-growing approach (SPOT) (Yang et al., 2001). The effect of additive white Gaussian noise is also investigated. PMID:12906242

  14. Enzyme Microarrays Assembled by Acoustic Dispensing Technology

    PubMed Central

    Wong, E. Y.; Diamond, S. L.

    2008-01-01

    Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Utilizing acoustic dispensing technology, nanodroplets containing 10 µM ATP (3 µCi/µL 32P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40 nL compartments (100 droplets/slide) for Pim1 (Proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium-complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 µM substrate. The microarray was incubated at 30°C (97% Rh) for 1.5 hr. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. Using phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 µM to 3 µM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165 and average coefficients of variation (CVs) for the assay were ~20%. CVs for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development. PMID:18616925

  15. Laser direct writing of biomolecule microarrays

    NASA Astrophysics Data System (ADS)

    Serra, P.; Fernández-Pradas, J. M.; Berthet, F. X.; Colina, M.; Elvira, J.; Morenza, J. L.

    Protein-based biosensors are highly efficient tools for protein detection and identification. The production of these devices requires the manipulation of tiny amounts of protein solutions in conditions preserving their biological properties. In this work, laser induced forward transfer (LIFT) was used for spotting an array of a purified bacterial antigen in order to check the viability of this technique for the production of protein microarrays. A pulsed Nd:YAG laser beam (355 nm wavelength, 10 ns pulse duration) was used to transfer droplets of a solution containing the Treponema pallidum 17 kDa protein antigen on a glass slide. Optical microscopy showed that a regular array of micrometric droplets could be precisely and uniformly spotted onto a solid substrate. Subsequently, it was proved that LIFT deposition of a T. pallidum 17 kDa antigen onto nylon-coated glass slides preserves its antigenic reactivity and diagnostic properties. These results support that LIFT is suitable for the production of protein microarrays and pave the way for future diagnostics applications.

  16. Fabrication and Use of MicroEnvironment microArrays (MEArrays)

    PubMed Central

    Lin, Chun-Han; Lee, Jonathan K.; LaBarge, Mark A.

    2012-01-01

    The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours. Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes. MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the

  17. Label-free detection repeatability of protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Dai, Jun; Li, Lin; Wang, JingYi; He, LiPing; Lu, HuiBin; Ruan, KangCheng; Jin, KuiJuan; Yang, GuoZhen

    2012-12-01

    We examine the repeatabilities of oblique-incidence reflectivity difference (OIRD) method for label-free detecting biological molecular interaction using protein microarrays. The experimental results show that the repeatabilities are the same in a given microarray or microarray-microarray and are consistent, indicating that OIRD is a promising label-free detection technique for biological microarrays.

  18. Increasing the Number of Thyroid Lesions Classes in Microarray Analysis Improves the Relevance of Diagnostic Markers

    PubMed Central

    Fontaine, Jean-Fred; Mirebeau-Prunier, Delphine; Raharijaona, Mahatsangy; Franc, Brigitte; Triau, Stephane; Rodien, Patrice; Goëau-Brissonniére, Olivier; Karayan-Tapon, Lucie; Mello, Marielle; Houlgatte, Rémi; Malthiery, Yves; Savagner, Frédérique

    2009-01-01

    Background Genetic markers for thyroid cancers identified by microarray analysis have offered limited predictive accuracy so far because of the few classes of thyroid lesions usually taken into account. To improve diagnostic relevance, we have simultaneously analyzed microarray data from six public datasets covering a total of 347 thyroid tissue samples representing 12 histological classes of follicular lesions and normal thyroid tissue. Our own dataset, containing about half the thyroid tissue samples, included all categories of thyroid lesions. Methodology/Principal Findings Classifier predictions were strongly affected by similarities between classes and by the number of classes in the training sets. In each dataset, sample prediction was improved by separating the samples into three groups according to class similarities. The cross-validation of differential genes revealed four clusters with functional enrichments. The analysis of six of these genes (APOD, APOE, CLGN, CRABP1, SDHA and TIMP1) in 49 new samples showed consistent gene and protein profiles with the class similarities observed. Focusing on four subclasses of follicular tumor, we explored the diagnostic potential of 12 selected markers (CASP10, CDH16, CLGN, CRABP1, HMGB2, ALPL2, ADAMTS2, CABIN1, ALDH1A3, USP13, NR2F2, KRTHB5) by real-time quantitative RT-PCR on 32 other new samples. The gene expression profiles of follicular tumors were examined with reference to the mutational status of the Pax8-PPARγ, TSHR, GNAS and NRAS genes. Conclusion/Significance We show that diagnostic tools defined on the basis of microarray data are more relevant when a large number of samples and tissue classes are used. Taking into account the relationships between the thyroid tumor pathologies, together with the main biological functions and pathways involved, improved the diagnostic accuracy of the samples. Our approach was particularly relevant for the classification of microfollicular adenomas. PMID:19893615

  19. 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

  20. Receptor and enzyme expression for prostanoid metabolism in colorectal cancer related to tumor tissue PGE2.

    PubMed

    Gustafsson, Annika; Andersson, Marianne; Lagerstedt, Kristina; Lönnroth, Christina; Nordgren, Svante; Lundholm, Kent

    2010-02-01

    Prostaglandins support progression of colorectal cancer by several mechanisms. This conclusion is based on epidemiological and drug intervention long-term studies or retrieved from animal and cell culture experiments. The aim of the present study was to map receptor and enzyme expression for prostanoid metabolism in the presence of high or low PGE2 content within colon cancer tissue at primary tumor operation and after short-term preoperative provision of non-steroidal anti-inflammatory drug (NSAID). Twenty-three unselected patients with colon cancer were randomly selected to receive indomethacin (NSAID) or sham treatment for 3 days before surgery. Normal colon and tumor tissue were collected at operation for RNA extraction. Tissue PGE2 levels were measured by radioimmunoassay. Gene expression was quantified by microarray and real-time PCR. COX-1 expression increased proportionally to COX-2 expression in colon cancer tissue from untreated patients. Indomethacin reduced PGE2 content in normal and tumor tissue with subsequently decreased IP, HPGD and PPARgamma receptor expression in both tumor and normal colon tissue, while subtype EP1-4 receptors were not significantly influenced by indomethacin treatment. MPGES-1 expression was not related to overall PGE2 content in tumor and colon tissue, but decreased significantly in normal tissue during indomethacin exposure. Reduction of tumor tissue PGE2 was related to significant alteration in expression of several hundred genes indicating decreased cell cycling and increased apoptosis during indomethacin treatment, probably related to upregulation of acute phase reactants in tumor tissue. Increased prostanoid activity in colon cancer tissue is related to cross-talk between tumor and stroma cells. PMID:20043083

  1. New insights about host response to smallpox using microarray data

    PubMed Central

    Esteves, Gustavo H; Simoes, Ana CQ; Souza, Estevao; Dias, Rodrigo A; Ospina, Raydonal; Venancio, Thiago M

    2007-01-01

    Background Smallpox is a lethal disease that was endemic in many parts of the world until eradicated by massive immunization. Due to its lethality, there are serious concerns about its use as a bioweapon. Here we analyze publicly available microarray data to further understand survival of smallpox infected macaques, using systems biology approaches. Our goal is to improve the knowledge about the progression of this disease. Results We used KEGG pathways annotations to define groups of genes (or modules), and subsequently compared them to macaque survival times. This technique provided additional insights about the host response to this disease, such as increased expression of the cytokines and ECM receptors in the individuals with higher survival times. These results could indicate that these gene groups could influence an effective response from the host to smallpox. Conclusion Macaques with higher survival times clearly express some specific pathways previously unidentified using regular gene-by-gene approaches. Our work also shows how third party analysis of public datasets can be important to support new hypotheses to relevant biological problems. PMID:17718913

  2. Experimental Approaches to Microarray Analysis of Tumor Samples

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

    2008-01-01

    Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has…

  3. Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

    PubMed Central

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G.; Chandler, Darrell P.

    2014-01-01

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice. PMID:24796567

  4. The Importance of Normalization on Large and Heterogeneous Microarray Datasets

    EPA Science Inventory

    DNA microarray technology is a powerful functional genomics tool increasingly used for investigating global gene expression in environmental studies. Microarrays can also be used in identifying biological networks, as they give insight on the complex gene-to-gene interactions, ne...

  5. Demonstrating a multi-drug resistant Mycobacterium tuberculosis amplification microarray.

    PubMed

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G; Chandler, Darrell P

    2014-04-25

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

  6. Screening and characterization of plant cell walls using carbohydrate microarrays.

    PubMed

    Sørensen, Iben; Willats, William G T

    2011-01-01

    Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall materials is dependent to a large extent on being able to analyse their fine structures. We have developed a suite of techniques based on microarrays probed with monoclonal antibodies with specificity for cell wall components, and here we present practical protocols for this type of analysis.

  7. Microarray analysis of Xenopus endoderm expressing Ptf1a

    PubMed Central

    Bilogan, Cassandra K.; Horb, Marko E.

    2012-01-01

    Pancreas specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this paper, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification. PMID:22815262

  8. An ultralow background substrate for protein microarray technology.

    PubMed

    Feng, Hui; Zhang, Qingyang; Ma, Hongwei; Zheng, Bo

    2015-08-21

    We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis. PMID:26134063

  9. cDNA microarray screening in food safety.

    PubMed

    Roy, Sashwati; Sen, Chandan K

    2006-04-01

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests.

  10. Finding dominant sets in microarray data.

    PubMed

    Fu, Xuping; Teng, Li; Li, Yao; Chen, Wenbin; Mao, Yumin; Shen, I-Fan; Xie, Yi

    2005-01-01

    Clustering allows us to extract groups of genes that are tightly coexpressed from Microarray data. In this paper, a new method DSF_Clust is developed to find dominant sets (clusters). We have preformed DSF_Clust on several gene expression datasets and given the evaluation with some criteria. The results showed that this approach could cluster dominant sets of good quality compared to kmeans method. DSF_Clust deals with three issues that have bedeviled clustering, some dominant sets being statistically determined in a significance level, predefining cluster structure being not required, and the quality of a dominant set being ensured. We have also applied this approach to analyze published data of yeast cell cycle gene expression and found some biologically meaningful gene groups to be dug out. Furthermore, DSF_Clust is a potentially good tool to search for putative regulatory signals.

  11. 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.

  12. Microarray analysis reveals altered circulating microRNA expression in mice infected with Coxsackievirus B3

    PubMed Central

    Sun, Chaoyu; Tong, Lei; Zhao, Wenran; Wang, Yan; Meng, Yuan; Lin, Lexun; Liu, Bingchen; Zhai, Yujia; Zhong, Zhaohua; Li, Xueqi

    2016-01-01

    Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection. PMID:27698715

  13. Microarray analysis reveals altered circulating microRNA expression in mice infected with Coxsackievirus B3

    PubMed Central

    Sun, Chaoyu; Tong, Lei; Zhao, Wenran; Wang, Yan; Meng, Yuan; Lin, Lexun; Liu, Bingchen; Zhai, Yujia; Zhong, Zhaohua; Li, Xueqi

    2016-01-01

    Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection.

  14. SAMMD: Staphylococcus aureus Microarray Meta-Database

    PubMed Central

    Nagarajan, Vijayaraj; Elasri, Mohamed O

    2007-01-01

    Background Staphylococcus aureus is an important human pathogen, causing a wide variety of diseases ranging from superficial skin infections to severe life threatening infections. S. aureus is one of the leading causes of nosocomial infections. Its ability to resist multiple antibiotics poses a growing public health problem. In order to understand the mechanism of pathogenesis of S. aureus, several global expression profiles have been developed. These transcriptional profiles included regulatory mutants of S. aureus and growth of wild type under different growth conditions. The abundance of these profiles has generated a large amount of data without a uniform annotation system to comprehensively examine them. We report the development of the Staphylococcus aureus Microarray meta-database (SAMMD) which includes data from all the published transcriptional profiles. SAMMD is a web-accessible database that helps users to perform a variety of analysis against and within the existing transcriptional profiles. Description SAMMD is a relational database that uses MySQL as the back end and PHP/JavaScript/DHTML as the front end. The database is normalized and consists of five tables, which holds information about gene annotations, regulated gene lists, experimental details, references, and other details. SAMMD data is collected from the peer-reviewed published articles. Data extraction and conversion was done using perl scripts while data entry was done through phpMyAdmin tool. The database is accessible via a web interface that contains several features such as a simple search by ORF ID, gene name, gene product name, advanced search using gene lists, comparing among datasets, browsing, downloading, statistics, and help. The database is licensed under General Public License (GPL). Conclusion SAMMD is hosted and available at . Currently there are over 9500 entries for regulated genes, from 67 microarray experiments. SAMMD will help staphylococcal scientists to analyze their

  15. Analysis of environmental transcriptomes by DNA microarrays.

    PubMed

    Parro, Víctor; Moreno-Paz, Mercedes; González-Toril, Elena

    2007-02-01

    In this work we investigated the correlations between global gene expression patterns and environmental parameters in natural ecosystems. We studied the preferential gene expression of the iron oxidizer bacterium Leptospirillum ferrooxidans to adapt its physiology to changes in the physicochemical parameters in its natural medium. Transcriptome analysis by DNA microarrays can proportionate an instant picture about the preferential gene expression between two different environmental samples. However, this type of analysis is very difficult and complex in natural ecosystems, mainly because of the broad biodiversity and multiple environmental parameters that may affect gene expression. The necessity of high-quality RNA preparations as well as complicated data analysis are also technological limitations. The low prokaryotic diversity of the extremely acidic and iron-rich waters of the Tinto River (Spain) ecosystem, where L. ferrooxidans is abundant, allows the opportunity to achieve global gene expression studies and to associate gene function with environmental parameters. We applied a total RNA amplification protocol validated previously for the amplification of the environmental transcriptome (meta-transcriptome). The meta-transcriptome of two sites from the Tinto River mainly differing in the salt and oxygen contents were amplified and analysed by a L. ferrooxidans DNA microarray. The results showed a clear preferential induction of genes involved in certain physicochemical parameters like: high salinity (ectAB, otsAB), low oxygen concentration (cydAB), iron uptake (fecA-exbBD-tonB), oxidative stress (carotenoid synthesis, oxyR, recG), potassium (kdpBAC) or phosphate concentrations (pstSCAB), etc. We conclude that specific gene expression patterns can be useful indicators for the physiological conditions in a defined ecosystem. Also, the upregulation of certain genes and operons reveals information about the environmental conditions (nutrient limitations, stresses

  16. Lipid Microarray Biosensor for Biotoxin Detection.

    SciTech Connect

    Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.; Edel, Joshua B.; Meyer, Grant D.; Craighead, Harold G.

    2006-05-01

    We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates by TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4

  17. Development and assessment of oligonucleotide microarrays for Atlantic salmon (Salmo salar L.).

    PubMed

    Krasnov, Aleksei; Timmerhaus, Gerrit; Afanasyev, Sergey; Jørgensen, Sven Martin

    2011-03-01

    The cDNA microarrays have played a major role in functional genomics of fish and contributed substantially to different areas of aquaculture research. However at present these platforms are gradually substituted with oligonucleotide microarrays (ONM), which represent the most cost-efficient, flexible, powerful and accurate tool for multiple gene expression profiling, especially in species with rich genomic resources. This paper describes the development and assessment of ONM platforms for Atlantic salmon. The process started with the establishment of a bioinformatic system, selection of a low redundancy set of nucleotide sequences providing coverage of transcriptomes of several fish species, their identification by protein products and annotations. Pilot experiments were performed to address issues that are essential for development of ONM: gene composition, quality assessment, hybridization success of homologous and heterologous probes, optimum numbers of spot replicates and processing, management and mining of gene expression data. Performance of microarrays was evaluated in two experiments with Atlantic salmon. Comparison of peripheral blood leukocytes with a mixture of other tissues was conducted for characterization of the leukocyte transcriptome. Analyses of salmon infected with different viral diseases identified virus-responsive genes that can be used as markers for diagnostics of infected status of fish. Data mining with functional annotations confirmed the relevance of these findings.

  18. ZODET: Software for the Identification, Analysis and Visualisation of Outlier Genes in Microarray Expression Data

    PubMed Central

    Roden, Daniel L.; Sewell, Gavin W.; Lobley, Anna; Levine, Adam P.; Smith, Andrew M.; Segal, Anthony W.

    2014-01-01

    Summary Complex human diseases can show significant heterogeneity between patients with the same phenotypic disorder. An outlier detection strategy was developed to identify variants at the level of gene transcription that are of potential biological and phenotypic importance. Here we describe a graphical software package (z-score outlier detection (ZODET)) that enables identification and visualisation of gross abnormalities in gene expression (outliers) in individuals, using whole genome microarray data. Mean and standard deviation of expression in a healthy control cohort is used to detect both over and under-expressed probes in individual test subjects. We compared the potential of ZODET to detect outlier genes in gene expression datasets with a previously described statistical method, gene tissue index (GTI), using a simulated expression dataset and a publicly available monocyte-derived macrophage microarray dataset. Taken together, these results support ZODET as a novel approach to identify outlier genes of potential pathogenic relevance in complex human diseases. The algorithm is implemented using R packages and Java. Availability The software is freely available from http://www.ucl.ac.uk/medicine/molecular-medicine/publications/microarray-outlier-analysis. PMID:24416128

  19. ONCOMINE: a cancer microarray database and integrated data-mining platform.

    PubMed

    Rhodes, Daniel R; Yu, Jianjun; Shanker, K; Deshpande, Nandan; Varambally, Radhika; Ghosh, Debashis; Barrette, Terrence; Pandey, Akhilesh; Chinnaiyan, Arul M

    2004-01-01

    DNA microarray technology has led to an explosion of oncogenomic analyses, generating a wealth of data and uncovering the complex gene expression patterns of cancer. Unfortunately, due to the lack of a unifying bioinformatic resource, the majority of these data sit stagnant and disjointed following publication, massively underutilized by the cancer research community. Here, we present ONCOMINE, a cancer microarray database and web-based data-mining platform aimed at facilitating discovery from genome-wide expression analyses. To date, ONCOMINE contains 65 gene expression datasets comprising nearly 48 million gene expression measurements form over 4700 microarray experiments. Differential expression analyses comparing most major types of cancer with respective normal tissues as well as a variety of cancer subtypes and clinical-based and pathology-based analyses are available for exploration. Data can be queried and visualized for a selected gene across all analyses or for multiple genes in a selected analysis. Furthermore, gene sets can be limited to clinically important annotations including secreted, kinase, membrane, and known gene-drug target pairs to facilitate the discovery of novel biomarkers and therapeutic targets.

  20. Microarray analysis of gene expression in disk abalone Haliotis discus discus after bacterial challenge.

    PubMed

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Lee, Youngdeuk; Whang, Ilson; Lee, Jae-Seong; Choi, Cheol Young; Lee, Jehee

    2011-02-01

    In this study, we investigated the gene expression profiling of disk abalone, Haliotis discus discus challenged by a mixture of thr