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.
Park
Jik Young
JY
Department of Pathology, College of Medicine, The Catholic University of Korea, #505 Banpo-dong Seocho-gu, Seoul 137-701, South Korea.
Kim
Su Young
SY
Lee
Jong Heun
JH
Song
Jaehwi
J
Noh
Ji Heon
JH
Lee
Sug Hyung
SH
Park
Won Sang
WS
Yoo
Nam Jin
NJ
Lee
Jung Young
JY
Nam
Suk Woo
SW
D018390:Gene Targeting / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D018075:RNA, Complementary / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018075:RNA, Complementary