Fig. 1
RNase If -qPCR assay overview. The total RNA is isolated first (a) using the protocol described in materials and methods. Then the isolated total RNAs are treated with RNase If and followed by RNA clean-up to obtain the purified dsRNA (loop sequence is digested) (b). The RNase If -treated RNAs are proceeded for cDNA conversion. The RNAs are pre-incubated with random hexamers at 95 °C (c) and 70 °C (d), respectively, and followed by cDNA conversion. Blue strands represent the reversely transcribed cDNA for the RNA incubated in 95 °C. In contrast, no cDNA is produced from the RNA incubated in 70 °C if all ssRNA is digested by RNase If. In addition, non-RNase If -treated RNA was used for cDNA conversion at 95 °C (e) and 70 °C (f). For RNA pre-incubated in 95 °C. cDNAs (dark blue strand) are transcribed from both dsRNA and ssRNA. Oppositely, cDNAs are only converted from ssRNAs (including RNAi and endogenous gene). The qRT-PCR is performed after the cDNA conversion using the custom designed TaqMan™ assay