Tomasello Luisa, Cluts Landon, Croce Carlo M
Department of Cancer Biology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2019;1970:341-353. doi: 10.1007/978-1-4939-9207-2_19.
MicroRNAs are a class of small noncoding RNA involved in the mechanism of RNA silencing and regulation of gene expression at a posttranscriptional level. Recently, the discovery of their targets led to the understanding of the molecular role of these small molecules and their involvement in the pathogenesis of numerous diseases, including cancer. Not long ago, the improvement of several informatics tools for microRNA target prediction has supported the experimental research through the selection of potential mRNA as microRNA target candidates. Since the regulation mediated by microRNA affects gene expression at a posttranscriptional level, the analysis of the proteins encoded by the gene targets is essential in understanding the involvement of these small molecules in biological processes and their role in several diseases. In this chapter, we describe the experimental procedure of Western blotting applied to the validation of microRNA targets. Western blotting is one of the most common and largest know technique for protein analysis. This method, coupled with the luciferase assay, represents the standard procedure for the experimental confirmation of microRNA targeting.
微小RNA是一类小的非编码RNA，参与RNA沉默机制以及转录后水平的基因表达调控。最近，对其靶标的发现使人们了解了这些小分子的分子作用及其在包括癌症在内的多种疾病发病机制中的作用。不久前，几种用于微小RNA靶标预测的信息学工具的改进，通过选择潜在的mRNA作为微小RNA靶标候选物，为实验研究提供了支持。由于微小RNA介导的调控在转录后水平影响基因表达，因此对基因靶标编码的蛋白质进行分析对于理解这些小分子在生物过程中的作用及其在多种疾病中的作用至关重要。在本章中，我们描述了用于验证微小RNA靶标的蛋白质印迹实验步骤。蛋白质印迹是最常用且广为人知的蛋白质分析技术之一。该方法与荧光素酶测定法相结合，是实验确认微小RNA靶向作用的标准程序。
