{"title":"Simultaneous In Situ Detection of m<sup>6</sup>A-Modified and Unmodified RNAs Using DART-FISH.","authors":"Charles J Sheehan, Kate D Meyer","doi":"10.1007/978-1-0716-3766-1_10","DOIUrl":null,"url":null,"abstract":"<p><p>N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m<sup>6</sup>A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m<sup>6</sup>A-modified and unmodified target transcripts. DART-FISH combines m<sup>6</sup>A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m<sup>6</sup>A stoichiometry and methylated mRNA localization in single cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2784 ","pages":"147-161"},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in molecular biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/978-1-0716-3766-1_10","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
N6-methyladenosine (m6A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m6A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m6A-modified and unmodified target transcripts. DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m6A stoichiometry and methylated mRNA localization in single cells.
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For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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