Microarray profiling is feasible using archived tissue from a Cooperative Group Clinical Trial: Results from a pilot study in CALGB 9342
Lyndsay N. HarrisCharles M. PerouZoltán SzállásiA. E. EklundS. CarterFengming YouGloria BroadwaterLaura MonovichEP WinerMark G. ErlanderMatthew J. Ellis
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545 Background: CALGB 9342 was designed to test the efficacy of paclitaxel in women with advanced breast cancer. Tissue blocks were collected to retrospectively assess single gene markers (Lin et al, ASCO 2004, Abstract # 9562). RNA profiling has been able to classify tumors into biologic subtypes: ER/PR positive, HER2 amplified and basal-like (ER/PR/HER2 negative) (Perou et al, Nature 2001). To assess the feasibility of performing RNA microarray from this archived material, a pilot study was performed to determine if tumor subtypes could be identified by gene expression profiles. Methods: Primary tumors diagnosed between 1990–97 from women with metastatic breast cancer enrolled on CALGB 9342 were selected. Thirty formalin fixed, paraffin-embedded (FFPE) tissue blocks were selected based on HER2 by FISH (Vysis Pathvysion.), estrogen and progesterone receptor status (from pathology report) to equally represent three categories of breast cancer: HER2 amplified, ER and/or PR positive, ER/PR/HER2 negative. 4 uM tissue sections were macrodissected, RNA was isolated, linearly amplified, hybridized to two microarray platforms (Custom Agilent 22K gene chip, Affymetrix X3P whole genome array) using appropriate labeling methods. Tumors were classified by gene expression pattern using intrinsic signature genes and the ability to predict biomarker status was subsequently determined. Results: Twenty-eight of 30 cases could be successfully amplified and hybridized for microarray profiling. Ten percent of Agilent arrays and 20% of Affymetrix arrays were of poor quality. Both Agilent Whole Genome Chip and the Affymetrix X3P array correctly classified 9/10 HER2 positive, 8/9 ER/PR positive and 9/9 triple negative cases. Neither poor quality nor misclassification could be explained by year of embedding of the primary tumor. Conclusions: Microarray profiling from archived, FFPE tissue from cooperative group clinical trials is able to correctly classify tumor subtypes in the majority of cases. This data suggests that microarray profiling from large cohorts of archived tissue may be feasible to search for gene expression patterns that predict outcome. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Arcturus ArcturusHierarchical clustering
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Microarray can parallel quantification of large numbers of genes and promises to provide detailed insight into cellular processes involved in the regulation of gene expression. In plants, microarray for gene expression profiling should provide an important way for understanding of gene functions and signaling networks that operate plants growth and development, respond to biotic and abiotic stresses, important agronomic characters. This review focuses on the application of microarray for gene expression profiling in plants. Moreover, development and application of tobacco microarray is also summarized. This review will provide useful information for better application of plant microarray in studies of gene function and regulation mechanism.
Microarray databases
Gene chip analysis
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Profiling (computer programming)
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Gene chip analysis
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OBJECTIVE To investigate the changes of gene expression profile of rat liver tissue by cDNA microarrays. METHODS Twenty Wistar rats in control group (n = 10) and isoniazid (INH) group (n = 10) were orally administrated with normal saline and 400 mg/kg INH for 14 days, respectively. The differentially expressed genes significantly correlated with liver injury were screened and analyzed. The mechanisms of liver injury caused by INH were specifically analyzed at level of gene expression based on the biological functions of those differentially expressed genes. RESULTS Thirty-seven differentially expressed genes were found in the rats administrated with INH. Among them, 25 genes were up-regulated, while the other 12 genes were down-regulated. These differentially expressed genes were functionally related to the changes of CYP450-related genes, fatty acid metabolism, and protein metabolism. CONCLUSION INH can cause the remarkable changes of the gene expression profiles in rat liver cells, which is important for further elucidating the mechanisms of liver injury caused by INH.
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Objective To identify the differentially expressed genes of osteoblasts under the stimulation of mechano growth factor E peptide(MGF-Ct24E) and mechanical stress by microarray analysis.Methods Primary osteoblasts were cultured in vitro,which were subjected to mechanical stimulation(with the mechanical strain of 12% and frequency of 0.5 Hz) and MGF-Ct24E treatment(50 mg/L),respectively.The gene expression profiles were analysed by cDNA microarrys and quantitative PCR was used to validate the microarray data.Results Compared with the control group,1 866 genes were found to have differentially expressed in the mechanical loading group,in which 1 113 genes were up-regulated,while 753 genes were down-regulated.1 178 genes were found to have differentially expressed in the MGF-Ct24E group,in which 796 genes were up-regulated and 382 genes were down-regulated.GO analysis suggested that the gene expression profile of MGF-Ct24E group was consistent with that of the mechanical loading group and differentially expressed genes were mainly involved in cell proliferation and differentiation,response to mechanical stress and mechaotransduction.Conclusions The microarray analysis showed that MGF-Ct24E treatment had similar effects with the mechanical loading on the gene expression of osteoblasts,which might provide a novel approach to study the usage of MGF-Ct24E for treating bone repair in the absence of mechanical stimulation.
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Aging and aging-related diseases are associated with altered patterns of gene expression, involving quantitative and qualitative changes in the abundance of specific transcripts. A complete and simultaneous analysis of gene expression should therefore lead to important insights into the transcriptional mechanisms underlying the aging process. Recently, we have employed high-throughput gene expression profiling to study transcriptional activity in heart. Two technologies, serial analysis of gene expression (SAGE) and gene expression arrays, allow rapid, large-scale expression profiling, which provides information about the dynamics of total gene expression with age and which can be employed to identify candidate genes that may serve as diagnostic and prognostic markers in age-associated cardiac diseases. The accompanying gene predictions from high-throughput gene expression profiling provide a starting point for understanding the function, the complexity of interactions, and the role of genes in promoting cellular/organismal phenotypes during senescence and disease. In this review we describe the current state of transcriptome profiling by SAGE and microarrays and discuss how results generated with these approaches in heart can be applied to the study of aging and the treatment of cardiovascular diseases.
Senescence
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Objective To investigate the estrogen receptor(ER)isoforms ERα and ERβ expressions and their relationship with clinicopathological parameters in normal breast tissue,benign breast diseases,breast cancer in situ and invasive breast cancer tissue.Methods Breast tissue samples were obtained,including normal breast tissue(14 cases),benign breast diseases(35 cases),breast cancer in situ(8 cases)and invasive breast cancer(37 cases).Immunohistochemistry was used to measure the expressions of ERα and ERβ in the breast tissues.Results The profiles of the ERα and ERβ expressions in normal tissues and the tissues of benign breast diseases were similar.Compared with normal breast tissues and benign breast diseases,ERα protein level was down-regulated in the tissues of breast cancer in situ and invasive breast cancer significantly(P0.01);ERβ protein level was down-regulated in the tissues of invasive breast cancer notably(P0.01);The coexpression of ERα and ERβ was decreased in the tissues of breast cancer in situ and invasive breast cancer remarkably(P0.01).Conclusion ERβ may play a role in the human breast cancer probably by modulating ERα activity.
Breast tissue
CA 15-3
Estrogen receptor beta
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Gamma radiation acts via the indirect effect to damage cells by producing reactive oxygen species (ROS). These ROS are capable damaging macromolecules and, altering signal pathways and gene transcription. Cells have evolved enzymes and mechanisms to scavenge ROS and repair oxidative damage. Microarrays allow the survey of the gene transcription activity of thousands of genes simultaneously. Messenger RNA is extracted from cells, hybridized with the complementary DNA (cDNA) of a microarray chip, and examined with a chip reader. Affymetrix microarray chips have been produced by the CSCHAH in Winnipeg containing 26000 murine genes. Groups of female mice have been exposed to low dose whole body chronic gamma radiation exposures of 0,50,100, and 120 mGy, corresponding to 15,30,60, and 75 weeks, respectively. MRNA from mice brain tissue has been extracted, isolated, converted to cDNA and labeled. Gene expression in each irradiated mouse was compared to the pooled expression of the control mice. Analysis of gene expression levels are performed with microarray analytical software, Array Pro by Media Cybernetics, and powerful statistical software, BRB microarray tools. Differences in gene expressions, focusing on genes for cytokines, DNA repair mechanisms, immuno‐modulators, apoptosis pathways, and enzymatic anti‐oxidant systems, are being examined and will be reported.
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Objective The pilot study was to detect gene expression profiles in schizophrenia using microarray analysis. Methods Leucocytes in peripheral blood of six patients and six normal controls were collected and total RNA was abstracted. The cDNA probes were prepared by labeling cell RNA with Cy3-dUTP and Cy5-dUTP with reverse transcription. The arrays with 8 464 genes were hybridized against the cDNA probe mixture,and the fluorescent signals were scanned. Results Thirteen genes were down-regulated in all schizophrenic subjects when the ratio of Cy5∶Cy3 signal was greater than 2.0 or smaller than 0.5;and 31 genes expression altered when greater than 1.5 or smaller than 0.7. Out of the 31 genes,29 genes showed decreased expression and 2 genes elevated expression in schizophrenics. Conclusions The results provide preliminary evidence that some genes involved in immune,neuronal development,myelination formation,neurotransmission may have different expressions in schizophrenia from normal people.
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