Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.
2'-O-methylation (Nm) is an RNA modification commonly found on rRNA and snRNA, and at the mRNA 5'-cap, but has more recently been found internally on mRNA. The study of internal Nm modifications on mRNA is in the early stages, but we have reported that this sort of Nm modification can regulate mRNA abundance and translation. Although there are many methods to determine the presence of Nm on rRNA, detecting Nm on specific mRNA transcripts is technically difficult because they are much less abundant than rRNA. Some of these methods rely on the fact that Nm modification of RNA disrupts reverse transcription reactions when performed at low dNTP concentrations. In this chapter, we describe our approach to using quantitative PCR in conjunction with reverse transcription at low dNTPs, which is sensitive enough to detect changes to Nm modification of mRNA.
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