摘要
2′-O-甲基化是存在于多种小RNA 3′末端的修饰,具有重要的生物学功能。目前高通量研究小RNA 3′末端2′-O-甲基化的方法主要是NaIO4氧化处理结合小RNA深度测序,但该方法需要3μg总RNA,难以用于研究少量细胞中小RNA的3′末端甲基化修饰。该研究通过调整NaIO4氧化处理的条件以及测试有无甘油终止氧化反应对小RNA文库构建的影响,优化了适用于微量RNA的氧化条件,实现对10 ng小鼠睾丸样本总RNA中的小RNA 3′末端甲基化修饰的深度测序检测,建立了用于检测微量RNA样品中小RNA 3′末端甲基化修饰的方法,为检测少量细胞中小RNA的3′末端甲基化修饰提供了有效方法。
The 2′-O-methylation is present at the 3′end of various small RNAs and has important biological functions.At present,NaIO4 oxidation treatment combined with deep sequencing is one of the most powerful method to study the 2′-O-methylation of small RNAs.However,currently used protocol requires 3μg of total RNA and is not suitable for studying 3′terminal 2′-O-methylation of small RNAs in limited number of cells.In this study,we optimized the oxidation condition and omitted the glycerol when stops the oxidation reaction.With these improvements,we are able to identify the small RNAs bearing 3′terminal 2′-O-methylation using as little as 10 ng total RNA from mouse testis,providing a robust protocol suitable for investigating 3′terminal 2′-O-methylation of small RNAs in a small number of cells.
作者
薛皖强
侯丽
李荣红
吴立刚
XUE Wanqiang;HOU Li;LI Ronghong;WU Ligang(School of Life Science,Shanghai University,Shanghai 200444,China;State Key Laboratory of Cell Biology,CAS Center for Excellence in Molecular Cell Science,Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,University of Chinese Academy of Sciences,Shanghai 200031,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2019年第9期1779-1786,共8页
Chinese Journal of Cell Biology
基金
国家重点研发计划(批准号:2017YFA0504400)资助的课题~~
