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Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples.

ABSTRACT: BACKGROUND: The Affymetrix GeneChip® system is a commonly used platform for microarray analysis but the technology is inherently expensive. Unfortunately, changes in experimental planning and execution, such as the unavailability of previously anticipated samples or a shift in research focus, may render significant numbers of pre-purchased GeneChip® microarrays unprocessed before their manufacturer's expiration dates. Researchers and microarray core facilities wonder whether expired microarrays are still useful for gene expression analysis. In addition, it was not clear whether the two human reference RNA samples established by the MAQC project in 2005 still maintained their transcriptome integrity over a period of four years. Experiments were conducted to answer these questions. RESULTS: Microarray data were generated in 2009 in three replicates for each of the two MAQC samples with either expired Affymetrix U133A or unexpired U133Plus2 microarrays. These results were compared with data obtained in 2005 on the U133Plus2 microarray. The percentage of overlap between the lists of differentially expressed genes (DEGs) from U133Plus2 microarray data generated in 2009 and in 2005 was 97.44%. While there was some degree of fold change compression in the expired U133A microarrays, the percentage of overlap between the lists of DEGs from the expired and unexpired microarrays was as high as 96.99%. Moreover, the microarray data generated using the expired U133A microarrays in 2009 were highly concordant with microarray and TaqMan® data generated by the MAQC project in 2005. CONCLUSIONS: Our results demonstrated that microarray data generated using U133A microarrays, which were more than four years past the manufacturer's expiration date, were highly specific and consistent with those from unexpired microarrays in identifying DEGs despite some appreciable fold change compression and decrease in sensitivity. Our data also suggested that the MAQC reference RNA samples, stored at -80°C, were stable over a time frame of at least four years.

SUBMITTER: Wen Z

PROVIDER: S-EPMC3026357 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples.

Wen Zhining Z Wang Charles C Shi Quan Q Huang Ying Y Su Zhenqiang Z Hong Huixiao H Tong Weida W Shi Leming L

BMC bioinformatics 20101007

<h4>Background</h4>The Affymetrix GeneChip® system is a commonly used platform for microarray analysis but the technology is inherently expensive. Unfortunately, changes in experimental planning and execution, such as the unavailability of previously anticipated samples or a shift in research focus, may render significant numbers of pre-purchased GeneChip® microarrays unprocessed before their manufacturer's expiration dates. Researchers and microarray core facilities wonder whether expired micro ...[more]

PMID: 20946593

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Project description:BACKGROUND: Microarrays are a powerful tool for transcriptome analysis. Best results are obtained using high-quality RNA samples for preparation and hybridization. Issues with RNA integrity can lead to low data quality and failure of the microarray experiment. RESULTS: Microarray intensity data contains information to estimate the RNA quality of the sample. We here study the interplay of the characteristics of RNA surface hybridization with the effects of partly truncated transcripts on probe intensity. The 3'/5' intensity gradient, the basis of microarray RNA quality measures, is shown to depend on the degree of competitive binding of specific and of non-specific targets to a particular probe, on the degree of saturation of the probes with bound transcripts and on the distance of the probe from the 3'-end of the transcript. Increasing degrees of non-specific hybridization or of saturation reduce the 3'/5' intensity gradient and if not taken into account, this leads to biased results in common quality measures for GeneChip arrays such as affyslope or the control probe intensity ratio. We also found that short probe sets near the 3'-end of the transcripts are prone to non-specific hybridization presumable because of inaccurate positional assignment and the existence of transcript isoforms with variable 3' UTRs. Poor RNA quality is associated with a decreased amount of RNA material hybridized on the array paralleled by a decreased total signal level. Additionally, it causes a gene-specific loss of signal due to the positional bias of transcript abundance which requires an individual, gene-specific correction. We propose a new RNA quality measure that considers the hybridization mode. Graphical characteristics are introduced allowing assessment of RNA quality of each single array ('tongs plot' and 'degradation hook'). Furthermore, we suggest a method to correct for effects of RNA degradation on microarray intensities. CONCLUSIONS: The presented RNA degradation measure has best correlation with the independent RNA integrity measure RIN, and therefore presents itself as a valuable tool for quality control and even for the study of RNA degradation. When RNA degradation effects are detected in microarray experiments, a correction of the induced bias in probe intensities is advised.

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Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples.

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Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples.

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