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 (rho = 0.708, p < 0.0001, n = 277) using these platforms. The contribution of factors including up- vs. down-regulation, spot intensity, rho-value, fold-change, cycle threshold (C-t), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and rho-value) proves to be the most critical factor, with significant correlations of rho > 0.80 consistently observed when quality scores are applied.