Abstract
BackgroundThe MicroArray Quality Control (MAQC) project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006). The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta) RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency.ResultsThe SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes.ConclusionThese data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.
Highlights
The MicroArray Quality Control (MAQC) project evaluated the inter- and intraplatform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006)
For cross-platform comparisons between SYBR Green PCR Arrays and other gene expression analysis platforms evaluated in the MAQC study, data from the other technologies were obtained from published results [8,12] and from the database accessible from the MAQC website [20]
Results obtained from SYBR Green PCR Arrays show that 83 out of the 90 selected genes (92%) are considered to be present in both samples A and B, which is similar to the values reported in the MAQC study for the detection sensitivity of the other three quantitative platforms [8]
Summary
The MicroArray Quality Control (MAQC) project evaluated the inter- and intraplatform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006). The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta) RT-PCRTM and QuantiGene®. Concerns regarding the reliability and consistency of the microarray technology from different suppliers, different test sites and when using different methods for data processing and normalization have been raised [3,4,5,6,7] To address those concerns, scientists from the US Food and Drug Administration (FDA) established the MicroArray Quality Control (MAQC) consortium to evaluate the performance of several microarray platforms as well as three quantitative gene expression assays [8,9,10,11,12,13].
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