mRAP，一种用于测定微小RNA表达谱的灵敏方法。

mRAP, a sensitive method for determination of microRNA expression profiles.

作者信息

Mano Hiroyuki, Takada Shuji

机构信息

Division of Functional Genomics, Jichi Medical University, 3311-1 Yakushiji, Shimotsukeshi, Tochigi 329-0498, Japan.

出版信息

Methods. 2007 Oct;43(2):118-22. doi: 10.1016/j.ymeth.2007.04.006.

DOI:10.1016/j.ymeth.2007.04.006

PMID:17889798

Abstract

MicroRNAs (miRNAs) are noncoding RNA molecules of 21-24 nucleotides that regulate the expression of target genes in a posttranscriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling strategy, termed miRNA amplification profiling (mRAP), that relies on the use of a long, optimized 5' adaptor, the SMART (switching mechanism at the 5' end of RNA templates of reverse transcriptase) method, the polymerase chain reaction, and cDNA concatamerization after BanI digestion. This approach is highly sensitive, readily allowing the isolation of > 1 x 10(4) independent miRNA-derived cDNAs from < or = 1 x 10(4) cells. The mRAP method thus makes it possible to analyze miRNA expression profiles for small quantities of tissue or cells such as fresh clinical specimens.

摘要

微小RNA（miRNA）是一类由21 - 24个核苷酸组成的非编码RNA分子，它们以转录后调控的方式调节靶基因的表达。尽管有证据表明miRNA在胚胎发育、细胞分化以及人类疾病的发病机制中发挥着重要作用，但由于缺乏灵敏的克隆方法，细胞或组织中广泛的miRNA分析受到了阻碍。在此，我们描述了一种高效的分析策略，称为miRNA扩增分析（mRAP），该策略依赖于使用一个长的、经过优化的5'接头、SMART（逆转录酶RNA模板5'端的切换机制）方法、聚合酶链反应以及BanI消化后的cDNA串联。这种方法高度灵敏，能够轻松地从≤1×10⁴个细胞中分离出>1×10⁴个独立的源自miRNA的cDNA。因此，mRAP方法使得分析少量组织或细胞（如新鲜临床标本）的miRNA表达谱成为可能。