Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
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DOI：
10.3791/3851
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发表时间：
2012-09-01
影响因子：
1.2
通讯作者：
Atasoy, Ulus
中科院分区：
文献类型：
--
作者：
Dahm, Garrett M.;Gubin, Matthew M.;Atasoy, Ulus
由于高通量测序和高效微阵列分析的发展，全局基因表达分析已成为一种简单且易于获得的数据收集形式。然而，在许多研究和疾病模型中，靶基因mRNA的稳态水平并不总是与稳态蛋白水平直接相关。转录后基因调控是两者之间差异的一个可能解释。在RNA结合蛋白（RBP）结合的驱动下，转录后调节通过与靶mRNA形成核糖核蛋白（RNP）复合物来影响mRNA的定位、稳定性和翻译。从RNP复合物中的细胞提取物中鉴定这些未知的从头mRNA靶标对于理解RBP的机制和功能及其对蛋白质输出的影响至关重要。该方案概述了一种称为RNP免疫沉淀-微阵列（RIP-芯片）的方法，该方法允许在不断变化的实验条件下识别核糖核蛋白复合物中相关的特定mRNA，沿着选项以进一步优化个体研究人员的实验。有了这个重要的实验工具，研究人员可以探索与转录后基因调控以及其他核糖核蛋白相互作用相关的复杂机制。
As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.