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{"title":"Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry","authors":"Ying Wu, Ya Ying Zheng, Qishan Lin, Jia Sheng","doi":"10.1002/cpnc.113","DOIUrl":null,"url":null,"abstract":"<p>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.</p><p><b>Basic Protocol 1</b>: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides</p><p><b>Basic Protocol 2</b>: Digestion of RNA samples extracted from cells</p><p><b>Basic Protocol 3</b>: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.113","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Nucleic Acid Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpnc.113","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Chemistry","Score":null,"Total":0}
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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
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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