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Method of isolating, labeling and profiling small RNAs

机译:分离,标记和分析小RNA的方法

摘要

Disclosed herein is a method of selectively labeling non-messenger RNA molecules by isolating total RNA from a tissue or cell, dissolving the isolated RNA, blocking the 3′ end of the RNA and adding T4 RNA ligase and a labeled nucleic acid adaptor. With the results that the T4 RNA ligase ligates the adaptor only to RNA having a 5′ phosphate group and only small RNA are labeled. A method of labeling the 5′ end of mRNA isolates total RNA from a tissue or cell, dissolving RNA in RNase-free water, removing a 5′ cap structure from the mRNA using tobacco acid pyrophosphatase (TAP), removing the TAP, blocking the 3′ end of the RNA molecules; and ligating an adaptor to the RNA by adding T4 RNA ligase and a labeled DNA or RNA adaptor. In another embodiment, there is disclosed a method of expression profiling small RNA by separating labeled RNA from capped RNA, providing a microarray comprising a plurality of probes hybridizable to small RNA, incubating the labeled small RNA with the microarray, washing unhybridized RNA from the microarray and drying the microarray, staining hybridized RNA on the microarray; and scanning the labeled microarray to determine the identity and quantity of labeling to the various miRNA probe sites and thus providing an expression profile of small RNA.
机译:本文公开了通过从组织或细胞中分离总RNA,溶解分离的RNA,封闭RNA的3'端并添加T4 RNA连接酶和标记的核酸衔接子来选择性标记非信使RNA分子的方法。结果是,T4 RNA连接酶仅将衔接子与具有5'磷酸基团的RNA连接,并且仅标记了小RNA。标记mRNA 5'端的方法是从组织或细胞中分离总RNA,将RNA溶解在无RNase的水中,使用烟草酸焦磷酸酶(TAP)从mRNA去除5'帽结构,去除TAP,阻断RNA分子的3'末端;通过添加T4 RNA连接酶和标记的DNA或RNA衔接子,将衔接子与RNA连接。在另一个实施方案中,公开了通过以下方法表达表达小RNA的方法:通过从封端的RNA分离标记的RNA,提供包含多个可与小RNA杂交的探针的微阵列,将标记的小RNA与微阵列一起孵育,从微阵列中洗涤未杂交的RNA。干燥微阵列,在微阵列上染色杂交的RNA。扫描标记的微阵列,以确定对各种miRNA探针位点标记的身份和数量,从而提供小RNA的表达谱。

著录项

  • 公开/公告号US2006246464A1

    专利类型

  • 公开/公告日2006-11-02

    原文格式PDF

  • 申请/专利权人 XUELIANG XIA;

    申请/专利号US20050116935

  • 发明设计人 XUELIANG XIA;

    申请日2005-04-28

  • 分类号C12Q1/68;C12P19/34;

  • 国家 US

  • 入库时间 2022-08-21 21:45:46

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