Application of amplified RNA and evaluation of cRNA targets for spotted-oligonucleotide microarray

Among different RNA amplification methods, T7 RNA polymerase-based in vitro transcription (IVT) that generates antisense RNA is most common in DNA microarray protocol. However, despite the fact that cRNA targets labeled during IVT are feasible for spotted-oligonucleotide microarray (spotted-oligoarray) hybridization due to complementary sequence of single-stranded oligonucleotide probe, no systemic assessment for the use of amplified cRNA targets has been reported for spotted-oligoarrays. In this investigation, we have compared the hybridization performance of amplified cRNA targets with that of cDNA targets from total RNA(T-RNA) using spotted-oligoarrays containing 18,864 genetic elements. Under the optimized hybridization conditions, we found that 86% of oligonucleotide probes were reproducibly detected by both cDNA and cRNA target protocols. In addition, cRNA targets generated by two-rounds of amplification of 10 ng T-RNA were concordant with first-round cRNA targets generated from 100 ng T-RNA by 0.858 of correlation coefficient. Taken together, we demonstrated that cRNA targets from very scant RNA amount could successfully be applied on spotted-oligoarrays, and hopefully this will facilitate the application of much smaller amount of source material based on the high-fidelity and improved target preparation of microarrays.