Jayaseelan Sabarinath, Doyle Francis, Tenenbaum Scott A
SUNY-College of Nanoscale Science and Engineering, Nanobioscience Constellation, State University of New York, Albany, NY 12203, USA.
SUNY-College of Nanoscale Science and Engineering, Nanobioscience Constellation, State University of New York, Albany, NY 12203, USA.
Methods. 2014 May 1;67(1):13-9. doi: 10.1016/j.ymeth.2013.11.001. Epub 2013 Nov 17.
Post-transcriptional regulation of messenger RNA contributes to numerous aspects of gene expression. The key component to this level of regulation is the interaction of RNA-binding proteins (RBPs) and their associated target mRNA. Splicing, stability, localization, translational efficiency, and alternate codon use are just some of the post-transcriptional processes regulated by RBPs. Central to our understanding of these processes is the need to characterize the network of RBP-mRNA associations and create a map of this functional post-transcriptional regulatory system. Here we provide a detailed methodology for mRNA isolation using RBP immunoprecipitation (RIP) as a primary partitioning approach followed by microarray (Chip) or next generation sequencing (NGS) analysis. We do this by using specific antibodies to target RBPs for the capture of associated RNA cargo. RIP-Chip/Seq has proven to be is a versatile, genomic technique that has been widely used to study endogenous RBP-RNA associations.
信使核糖核酸（mRNA）的转录后调控作用于基因表达的多个方面。这一调控水平的关键组成部分是RNA结合蛋白（RBPs）与其相关靶mRNA的相互作用。剪接、稳定性、定位、翻译效率以及密码子的交替使用只是受RBPs调控的转录后过程中的一部分。我们理解这些过程的核心在于需要对RBP-mRNA关联网络进行表征，并绘制出这个功能性转录后调控系统的图谱。在此，我们提供一种详细的方法，该方法以RBP免疫沉淀（RIP）作为主要的分离方法来分离mRNA，随后进行微阵列（芯片）或下一代测序（NGS）分析。我们通过使用特异性抗体靶向RBPs来捕获相关的RNA负载来实现这一目的。RIP-芯片/测序已被证明是一种通用的基因组技术，已广泛用于研究内源性RBP-RNA关联。
