Zhang Nannan, Hu Guowu, Myers Timothy G, Williamson Peter R
Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland.
Genomic Technologies Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Curr Protoc Immunol. 2019 Sep;126(1):e78. doi: 10.1002/cpim.78.
MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both in vitro and in vivo. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley & Sons, Inc.
微小RNA（miRNA）是短链（19至25个核苷酸）的非编码RNA分子，其靶向mRNA以抑制基因表达，并在调节许多基本生物学功能（包括细胞分化、发育、生长和代谢）中发挥重要作用。它们在真核细胞中高度保守，被认为是基因调控的重要古老元件。miRNA基因由RNA聚合酶II转录产生初级miRNA（pri-miRNA），其在细胞核中被微处理器复合物切割以产生称为前体miRNA（pre-miRNA）的茎环结构。前体miRNA被转运到细胞质中并被Dicer切割形成成熟的miRNA，后者通过加载到RNA诱导沉默复合体（RISC）并与靶mRNA内的互补序列结合，通过mRNA降解和/或翻译抑制来介导mRNA降解并抑制其翻译。由于人类基因组中报道了约1900个miRNA基因，其中许多与疾病相关，因此在生理和病理条件下研究miRNA表达和调控的合适方法对于包括免疫调节在内的人类生物学许多方面的研究变得越来越重要。与小干扰RNA（siRNA）一样，miRNA介导的靶向机制已被用于开发基于miRNA的疗法。为了进行完整而系统的分析，利用各种不同工具分析pri-mRNA、前体miRNA和成熟miRNA的表达并在体外和体内表征其靶标至关重要。此类研究将促进未来新型药物的设计和开发。本单元提供了六种miRNA分析的基本方案，涵盖下一代测序、定量实时PCR（qRT-PCR）以及基于地高辛的pri-mRNA、前体miRNA和成熟miRNA的表达分析；通过cDNA末端快速扩增（RACE）对pri-miRNA及其切割位点进行定位；电泳迁移率变动分析（EMSA）或基于生物素的miRNA-蛋白质复合物（miRNP）非放射性检测；以及使用miRNA模拟物和抑制剂对miRNA进行功能分析。©2019约翰威立父子公司。
