Wu Ligang, Belasco Joel G
Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, USA.
Methods Enzymol. 2008;449:373-93. doi: 10.1016/S0076-6879(08)02418-X.
MicroRNAs (miRNAs) utilize multiple posttranscriptional mechanisms to downregulate gene expression in metazoan organisms. These include translation repression and accelerated mRNA decay, the latter being triggered either by deadenylation or, less frequently, by endonucleolytic cleavage, as governed by the degree of complementarity of the targeted message. This chapter describes methods for examining the effect of miRNAs on the translation and turnover of complementary mRNAs in cultured mammalian cells. Among these are procedures for quantifying their influence on the cytoplasmic concentration and translation efficiency of luciferase reporter mRNAs, for monitoring their impact on the deadenylation and decay of beta-globin reporter mRNAs, and for detecting miRNA-directed internal mRNA cleavage.
微小RNA（miRNA）利用多种转录后机制下调后生动物中的基因表达。这些机制包括翻译抑制和加速mRNA降解，后者由去腺苷酸化触发，或者较少情况下由内切核酸酶切割触发，这取决于靶向mRNA的互补程度。本章介绍了检测miRNA对培养的哺乳动物细胞中互补mRNA的翻译和周转影响的方法。其中包括定量它们对荧光素酶报告基因mRNA的细胞质浓度和翻译效率影响的程序、监测它们对β-珠蛋白报告基因mRNA的去腺苷酸化和降解影响的程序，以及检测miRNA介导的mRNA内部切割的程序。
