Vatolin Sergei, Weil Robert J
San Francisco Laboratory CUBRC Inc, 670, San Francisco, California, USA.
Methods Enzymol. 2008;449:357-71. doi: 10.1016/S0076-6879(08)02417-8.
Mammalian cells express a large number of small, noncoding RNAs, including micro-RNAs (miRNAs), that can regulate both the level of a target mRNA and the protein produced by the target mRNA. Recognition of miRNA targets is a complicated process, as a single target mRNA may be regulated by several miRNAs. The potential for combinatorial miRNA-mediated regulation of miRNA targets complicates diagnostic and therapeutic applications of miRNAs. Despite significant progress in understanding the biology of miRNAs and advances in computational predictions of miRNA targets, methods that permit direct physical identification of miRNA-mRNA complexes in eukaryotic cells are still required. Several groups have utilized coimmunoprecipitation of RNA associated with a protein(s) that is part of the RNA silencing macromolecular complex. This chapter describes a detailed but straightforward strategy that identifies miRNA targets based on the assumption that small RNAs base paired with a complementary target mRNA can be used as a primer to synthesize cDNA that may be used for cloning, identification, and functional analysis.
哺乳动物细胞表达大量小的非编码RNA，包括微小RNA（miRNA），它们可以调节靶标mRNA的水平以及靶标mRNA产生的蛋白质。miRNA靶标的识别是一个复杂的过程，因为单个靶标mRNA可能受几种miRNA的调节。miRNA对靶标的组合介导调节的可能性使miRNA的诊断和治疗应用变得复杂。尽管在理解miRNA生物学方面取得了重大进展，并且在miRNA靶标的计算预测方面有所进步，但仍需要能够直接在真核细胞中物理鉴定miRNA-mRNA复合物的方法。有几个研究小组利用与作为RNA沉默大分子复合物一部分的一种或多种蛋白质相关的RNA进行共免疫沉淀。本章描述了一种详细但直接的策略，该策略基于这样的假设来鉴定miRNA靶标：与互补靶标mRNA碱基配对的小RNA可以用作引物来合成可用于克隆、鉴定和功能分析的cDNA。
