TY - JOUR

T1 - RNA immunoprecipitation to determine RNA-protein associations in vivo

AU - Selth, Luke A.

AU - Gilbert, Chris

AU - Svejstrup, Jesper Q.

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1101/pdb.prot5234

DO - 10.1101/pdb.prot5234

M3 - Journal article

AN - SCOPUS:68349098404

VL - 4

JO - Cold Spring Harbor Protocols

JF - Cold Spring Harbor Protocols

SN - 1940-3402

IS - 6

ER -