RNP-IP (Modified Method)-Getting Majority of RNA from RNA Binding Protein in Cytoplasm

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Post-transcriptional regulation of gene expression is a ribonucleoprotein (RNP)-driven process, which involves RNA binding proteins (RBPs) and noncoding RNAs that regulate splicing, nuclear export, subcellular localization, mRNA stability and translation. mRNAs encoding proteins that function in a particular cell process or pathway can be found within a unique mRNP complex, which consists of mRNA and RNP. This provides valuable information regarding not only known components of a particular process or pathway, but importantly, leads to the identification of novel components representing potential therapeutic targets and biomarkers. In addition to those targets identified by pathway expansion, the specific RBPs (RNA binding proteina) regulating RNA functions may be potential therapeutic targets in their own right. RNP-IP is a technology that allows the isolation and identification of mRNAs, microRNAs and protein components of RNP complexes from cell extracts using antibodies to RBPs. Once purified, the RNAs present in the complex are analyzed to identify the target mRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on microarray technology (chip analysis), or sequencing. Using this modified method will get more RNA existing in cytoplasm. This method does not require a pre-clear step and getting the supernatant for western blot is different from the original method., 基因表达的转录后调节是核糖核蛋白（RNP）驱动的过程，其涉及调节剪接，核出口，亚细胞定位，mRNA稳定性和翻译的RNA结合蛋白（RBP）和非编码RNA。编码在特定细胞过程或途径中起作用的蛋白质的mRNA可在由mRNA和RNP组成的独特mRNP复合物内发现。这不仅提供了关于特定过程或途径的已知组分的有价值信息，而且重要的是导致鉴定代表潜在治疗靶标和生物标志物的新组分。除了通过途径扩增鉴定的那些靶标之外，调节RNA功能的特异性RBP（RNA结合蛋白）可以是它们自己的潜在治疗靶标。 RNP-IP是一种允许使用针对RBP的抗体从细胞提取物中分离和鉴定RNP复合物的mRNA，微小RNA和蛋白质组分的技术。一旦纯化，使用各种分子生物学工具例如RT-PCR，基于微阵列技术（芯片分析）或测序的基因表达分析，分析复合物中存在的RNA以鉴定靶mRNA。使用这种修改的方法将获得更多的RNA存在于细胞质中。该方法不需要预清除步骤，并且获得用于western印迹的上清液不同于原始方法。