Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.
Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.
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DOI：
10.1007/978-1-0716-1374-0_11
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Holley CL
中科院分区：
文献类型：
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作者：
Elliott BA;Holley CL
2'-O-甲基化 (Nm) 是一种 RNA 修饰，常见于 rRNA、snRNA 和 mRNA 5'-帽上，但最近在 mRNA 内部发现。 mRNA内部Nm修饰的研究还处于早期阶段，但我们已经报道这种Nm修饰可以调节mRNA的丰度和翻译。尽管有很多方法可以确定 rRNA 上 Nm 的存在，但检测特定 mRNA 转录本上的 Nm 在技术上很困难，因为它们的丰度远低于 rRNA。其中一些方法依赖于以下事实：当在低 dNTP 浓度下进行时，RNA 的 Nm 修饰会破坏逆转录反应。在本章中，我们描述了在低 dNTP 下结合使用定量 PCR 和逆转录的方法，该方法足够灵敏，可以检测 mRNA Nm 修饰的变化。
2’-O-methylation (Nm) is an RNA modification commonly found on rRNA, 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.