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{"title":"质谱法检测和定量RNA硫代修饰","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":"{\"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}","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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