RT Journal Article
SR Electronic
T1 Site-specific validation and quantification of RNA 2′-O-methylation by qPCR with RNase H
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.05.23.493005
DO 10.1101/2022.05.23.493005
A1 Yifan Wu
A1 Yao Tang
A1 Yong Li
A1 Xiangwen Gu
A1 Qiang Wang
A1 Qihan Chen
YR 2022
UL http://biorxiv.org/content/early/2022/05/23/2022.05.23.493005.abstract
AB RNA 2′-O-methylation, one of the most abundant modifications on RNAs, is crucial for diverse intracellular biological processes. In the past several years, several high-throughput screening methods have been developed, resulting in the identification of thousands of new 2′-O-methylation (Nm) sites. However, due to the high variability in these high-throughput methods, accurate and rapid low-throughput validation assays are needed to confirm and quantify the 2′-O-methylation status of screened candidate sites. Although several low-throughput Nm site detection methods have been reported, precise location and quantitative assays are still challenging to achieve. Based on the characteristic that RNase H would be inhibited by Nm modification, we developed Nm-VAQ (site-specific 2′-O-methylation (Nm) Validation and Absolute Quantification resolution). In this study, with multiple tests of reagents and conditions, Nm-VAQ was established with a chimera probe of RNA/DNA, RNase H site-specific cleavage, and qRT-PCR, which demonstrated precise absolute quantification of modification ratios and methylation copy numbers. With the help of Nm-VAQ, the 2′-O-methylation status of 5 sites in rRNA was evaluated.Competing Interest StatementThe authors have declared no competing interest.