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Effect of long-term storage in TRIzol on microarray-based gene expression profiling.

ABSTRACT: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription.GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the "probe level" (a single consensus value) or "bead level" (multiple measurements provided by individual beads).TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were <2-fold changed. There was no consistent evidence that TRIzol causes premature reverse transcription termination. Instead, a subset of DEPs were systematically due to increased signals in TRIzol-preserved samples from probes near the 5' end of transcripts, suggesting better mRNA preservation with TRIzol.TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts.The standard practice of prolonged sample storage in TRIzol is suitable for GEP.

SUBMITTER: Ma W

PROVIDER: S-EPMC2966770 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Effect of long-term storage in TRIzol on microarray-based gene expression profiling.

Ma Wencai W Wang Michael M Wang Zhi-Qiang ZQ Sun Luhong L Graber David D Matthews Jairo J Champlin Richard R Yi Qing Q Orlowski Robert Z RZ Kwak Larry W LW Weber Donna M DM Thomas Sheeba K SK Shah Jatin J Kornblau Steven S Davis Richard E RE

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 20100830 10

<h4>Background</h4>Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription.<h4>Methods</h4>GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the "probe level" (a single consensus ...[more]

PMID: 20805315

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Project description:Renal cell carcinoma (RCC) is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays.Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC.Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR). Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved.This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most notably, genes involved in cell adhesion were dominantly up-regulated whereas genes involved in transport were dominantly down-regulated. This study reveals significant gene expression alterations in key biological pathways and provides potential insights into understanding the molecular mechanism of renal cell carcinogenesis.

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Effect of long-term storage in TRIzol on microarray-based gene expression profiling.

Publications

Effect of long-term storage in TRIzol on microarray-based gene expression profiling.

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