RIP （RNA-Binding Protein Immunoprecipitaiton）-A Tool to Explore Post-Transcriptional Regulation of RNAs
Gene expression plays an important role in complex cellular processes such as development, differentiation, and cellular response to environmental changes and is continuously modulated through a series of finely tuned post-transcriptional processes. Many of these processes utilize messengerRNA binding proteins （RBPs） to regulate the translation of functionally related gene products by binding subsets of mRNAs in a manner similar to how transcription factors regulate gene expression. While the regulation of gene expression by transcription factors has been well documented over time the regulation of gene products by RBPs is still new and has yet to be fully understood.
RNA-Binding protein immunoprecipitaiton （RIP） is the RNA analog of the more well-known ChIP application （chromatin immunoprecipitation）， which identifies DNA targets of DNA-binding proteins in an in-vivo cellular context. RIP can be used to identify specific RNA molecules （of many types） associated with specific nuclear or cytoplasmic proteins. These experiments involve immunoprecipitation of endogenously formed complexes of RNA binding proteins and co-isolation of any RNA species associated with that RNA binding protein. Purification of these RNA species allows interrogation and identification of mRNAs （and potentially non-coding RNAs associated with them） and can be directly subjected to a variety of applications including quantitative RT-PCR, microarray analysis （RIP-chip） and “deep-sequencing” or 2nd-generation sequencing based platform （RIP-Seq）.
To enable the adoption of these complex RIP methods we have focused on optimizing the methods and developing a variety of RIP validated reagents. Through this optimization process we have identified a number of key lessons that are critical for success and hope to share that in this discussion. By combining this learning with validated reagents we aim to provide researchers with a means of comparing their own results with the publicly available RNA profiles deposited by the ENCODE consortium.
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