Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry

Q4 Chemistry

Current Protocols in Nucleic Acid Chemistry Pub Date : 2020-08-21 DOI:10.1002/cpnc.113

Ying Wu, Ya Ying Zheng, Qishan Lin, Jia Sheng

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Abstract

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid-phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed-phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC-MS and LC-MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC-MS is employed first to identify RNA phosphorothioate modifications. An optimized LC-MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides

Basic Protocol 2: Digestion of RNA samples extracted from cells

Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry

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质谱法检测和定量RNA硫代修饰

本文描述了一种检测和定量细胞RNA样品中RNA硫代修饰的方法。从固相合成硫代磷酸核糖核酸二核苷酸开始，反相高效液相色谱纯化，制备硫代磷酸核糖核酸二核苷酸标准品，用于UPLC-MS和LC-MS/MS方法。使用TRIzol试剂从细胞中提取RNA样品，然后用核酸酶混合物消化，并用质谱法分析。UPLC-MS首先用于鉴定RNA磷酸化修饰。然后采用优化的LC-MS/MS方法量化一系列模型细胞中RNA硫代修饰的频率。©2020 Wiley期刊有限公司基本方案1:合成、纯化和表征RNA硫代二核苷酸基本方案2:从细胞中提取的RNA样品的消化基本方案3:通过质谱法检测和定量RNA硫代修饰

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Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry

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期刊介绍： Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.