Three-color cDNA microarrays with prehybridization quality control yield gene expression data comparable to that of commercial platforms.

Abstract

Despite their lower cost and high content flexibility, a limitation of in-house-prepared arrays has been their susceptibility to quality control (QC) issues and lack of QC standards across laboratories. Therefore, we developed a novel three-color array system that allows prehybridization QC as well as the Matarray software to facilitate acquisition of accurate gene expression data. In this study, we compared performance of our rat cDNA array to the Affymetrix RG-U34A and Agilent G4130A arrays using 2,824 UniGenes represented on all three arrays. Before data filtering, poor interplatform agreement was observed; however, after data filtering, differentially expressed UniGenes exhibited correlation coefficients of 0.91, 0.88, and 0.92 between the Affymetrix vs. Agilent, Affymetrix vs. cDNA, and Agilent vs. cDNA arrays, respectively. The Affymetrix, Agilent, and cDNA arrays agreed well with quantitative RT-PCR conducted on 42 UniGenes, yielding correlation coefficients of 0.90, 0.90, and 0.96, respectively. Each platform underestimated ratios relative to quantitative RT-PCR, possessing respective slopes of 0.86 (R2 = 0.81), 0.65 (R2 = 0.81), and 0.70 (R2 = 0.92). Overall, these data show that the combination of our novel technical and analytic approaches yield an accurate platform for functional genomics that is concordant with commercial discovery arrays in terms of identifying regulated genes and pathways.