Department of Medicine, Duke University Medical Center, Durham, NC, USA.
Methods Mol Biol. 2021;2298:171-184. doi: 10.1007/978-1-0716-1374-0_11.
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.
2'-O-甲基化（Nm）是一种常见的 RNA 修饰，存在于 rRNA 和 snRNA 以及 mRNA 的 5'-帽结构中，但最近也在内源 mRNA 上被发现。对 mRNA 内部 Nm 修饰的研究仍处于早期阶段，但我们已经报道过这种 Nm 修饰可以调节 mRNA 的丰度和翻译。尽管有许多方法可以确定 rRNA 上 Nm 的存在，但检测特定 mRNA 转录本上的 Nm 修饰在技术上具有挑战性，因为它们的丰度远低于 rRNA。其中一些方法依赖于这样一个事实，即在低 dNTP 浓度下进行 RNA 的反转录反应时，Nm 修饰会破坏反转录反应。在本章中，我们描述了一种使用定量 PCR 结合低 dNTP 反转录的方法，该方法足够灵敏，可以检测到 mRNA Nm 修饰的变化。
