Comparison of Two Different Amplification Methods for Small Amounts of Total RNA

Abstract: Two RNA amplification methods of in vitro transcription (IVT) and single primer amplification (WT-ovation) were compared to find a more efficient method for amplification of small amounts of total RNA. RNA was extracted from mouse cerebral cortex tissue or the layer V of cortical cortex， and then was amplified separately with the two different methods. After cDNA was reverse transcripted from the amplified RNA， real-time quantitative PCR was performed. The efficiency of RNA amplification was determined according to the results of real-time PCR. Our results indicated that WT-ovation amplification was about 2.8 times more efficient than IVT amplification. The value of D-Ct was positively correlated with both the primer length from the 3’ terminus of mRNA(P<0.05) and mRNA length(P<0.05) after IVT amplification. The nearer distance between primer and the 3’ terminus of mRNA and the shorter mRNA could result in lower value of D-Ct. But there was no significant correlation between the value of D-Ct and the primer position or mRNA length when the amplification method of WTovation was performed. Compared with IVT， WT-ovation was a more stable method which was more efficient and less affected by amplification factors.

CSTR: 32200.14.cjcb.2011.04.0007