Abstract
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.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from the NBL RIP-Assay Kit (see Reference 1).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.