Evaluation of amplified cRNA targets for oligonucleotide microarrays

Due to their hybridization specificity and capacity for systematic gene discovery, oligonucleotide-based microarray platforms offer numerous advantages over the cDNA microarrays currently widely used for comprehensive analysis of gene expression. Although fluorescently labeled amplified cRNA generated by T7 transcription is generally used in oligonucleotide microarrays, the feasibility of this combination (and that of cDNA microarrays) is yet to be studied systematically. In this paper, we performed a comparative study using a direct labeling method and T7 amplification to evaluate amplified cRNA targets for oligonucleotide microarrays. The efficiency of incorporation of Cy3- and Cy5-CTP into the target preparations, the reproducibility and the number of genes detected were investigated for each labeling approach and compared. The 12 genes that showed different expression profiles in the two labeling methods were evaluated by quantitative real-time PCR. In the 60-mer oligonucleotide microarray, amplified cRNA targets prepared by the T7 amplification method showed higher reproducibility and reliability than targets prepared by the direct labeling method in a comparative analysis of gene expression. This result also suggests the importance of fragmenting cRNA down to lengths of 50-200 bases before the hybridization process.