Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up — vs. down-regulation, spot intensity, ρ-value, fold-change, cycle threshold (Ct), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and ρ-value) proves to be the most critical factor, with significant correlations of ρτ;0.80 consistently observed when quality scores are applied.