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|Title:||RNA immunoprecipitation to determine RNA-protein associations in vivo|
|Citation:||Cold Spring Harbor Protocols, 2009; 4(6):1-7|
|Publisher:||Cold Spring Harbor Laboratory Press|
|Luke A. Selth, Chris Gilbert and Jesper Q. Svejstrup|
|Abstract:||The crucial roles of RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of mRNAs and subsequent control of gene expression patterns, have become increasingly evident. RNA immunoprecipitation (RIP) is a powerful technique used to detect the association of individual proteins with specific RNA molecules in vivo. Live cells are treated with formaldehyde to generate protein-RNA cross-links between proximal molecules. Following immunoprecipitation of a protein of interest and cross-link reversal, associated RNAs can be recovered, characterized, and quantitated by reverse transcriptase polymerase chain reaction (RT-PCR). Protein association with specific RNA regions can be performed under a variety of conditions (e.g., different environmental and cell-cycle states) and/or in mutant strains. Furthermore, because formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, RIP provides snapshots of protein-RNA interactions at specific time points and hence is useful for kinetic analyses of events occurring on RNA in vivo. The basics of RIP are very similar to chromatin immunoprecipitation (ChIP), but with some caveats that are important to appreciate to take full advantage of the possibilities afforded by RIP.|
|Keywords:||Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; RNA, Fungal; Cross-Linking Reagents; Immunoprecipitation; Protein Binding|
|Rights:||© 2009 Cold Spring Harbor Laboratory Press|
|Appears in Collections:||Agriculture, Food and Wine publications|
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