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Best practices for validating microarray expression data using VeriQuest®
qPCR Master Mixes from Affymetrix
Real-time qPCR is routinely used to provide inter-platform concordance to microarray results due to its high sensitivity and wide dynamic range. Affymetrix®
scientists measured gene expression levels by using the First-Strand cDNA Synthesis Kit
for Real-Time PCR [USB PN 75780] followed by VeriQuest Probe qPCR Master Mix [USB PN 75650] and compared these to the Affymetrix GeneChip®
HG-U133 Plus 2.0 dataset generated in the classic Microarray Quality Control (MAQC) study. Our results demonstrate, definitively, that VeriQuest qPCR Master Mixes can be used to validate Affymetrix microarray data.
MAQC Comparison The MAQC project correlated gene expression measurements from seven microarray and three alternative technology platforms for two reference RNA samples, MAQC A and MAQC B. The study included Affymetrix GeneChip HG-U133 Plus 2.0 array data from three test sites, and validated these with ABI TaqMan® were broadly expressed across all microarray platforms.1
assays performed on a subset of genes that Herein, we used 84 genes from the HG-U133 Plus 2.0 and ABI
TaqMan validated dataset where the expression in MAQC A vs. MAQC B RNA spans a wide range of fold-changes. Gene expression was measured using VeriQuest Probe qPCR Master Mix and both IDT PrimeTime® and was compared to the published MAQC results.
qPCR Primers and Probes,
Improved Results We show better concordance of real-time PCR expression results to Affymetrix GeneChip HG-U133 Plus 2.0 data with VeriQuest compared to ABI TaqMan. We further show that real-time PCR primer locations along the transcript do not significantly affect correlation to microar- ray data. We conclude that Affymetrix real-time PCR reagents can be reliably and successfully used to validate Affymetrix microarray data.
Fig. 1. Better correlations and slopes are achieved using VeriQuest qPCR master mix A.
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Site 1 R=0.94, Slope=0.59 Site 2 R=0.93, Slope=0.58 Site 3 R=0.94, Slope=0.61
-4 0 4 8 12 16 ABI TaqMan log2 FC Primers mostly towards 5' end C.
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Site 1 R=0.95, Slope=0.64 Site 2 R=0.95, Slope=0.63 Site 3 R=0.96, Slope=0.67
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qPCR Fold-change Correlation vs. Affymetrix HG-U133 Plus 2.0: Scatter plots compare the differential expression values (MAQC A vs. MAQC B) from three microarray test sites relative to values obtained by A) ABI TaqMan or B, C) VeriQuest qPCR master mix. qPCR was performed using VeriQuest Probe qPCR Master Mix with either B) primers mostly towards the 5’-end or C) primers mostly towards the 3’-end of the genes. The line shown is the linear best-fit to Site 1 data. Correlation coefficient (R) and Slope are listed for each site. Results reveal better correlation, using VeriQuest qPCR master mix, as summarized in Table 1.
Site 1 R=0.95, Slope=0.66 Site 2 R=0.96, Slope=0.65 Site 3 R=0.96, Slope=0.69
-4 0 4 8 12 16 USB VeriQuest log2 FC
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HG-U133 Plus 2.0 log2 FC
HG-U133 Plus 2.0 log2 FC
HG-U133 Plus 2.0 log2 FC
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