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https://hdl.handle.net/2440/54418
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Type: | Book chapter |
Title: | CLIP: cross-linking and immunoprecipitation of in vivo RNA targets of RNA binding proteins |
Author: | Jensen, K. Darnell, R. |
Citation: | RNA-Protein Interaction Protocols, 2008 / Haynes, S. (ed./s), vol.488, pp.85-98 |
Publisher: | Humana Press |
Publisher Place: | USA |
Issue Date: | 2008 |
Series/Report no.: | Methods in molecular biology ; v. 488 |
ISBN: | 9781588294197 |
Editor: | Haynes, S. |
Statement of Responsibility: | Kirk B. Jensen, Robert B. Darnell |
Abstract: | We present a newly developed method for fixing RNA-protein complexes in situ in living cells and the subsequent purification of the RNA targets. Using this approach, complex tissue such as mouse brain can be ultraviolet (UV) irradiated to covalently crosslink RNA-protein complexes. Once covalently bound, RNA-protein complexes can be purified under stringent conditions, allowing a highly specific purification scheme to be employed. After UV irradiation, the tissue is solubilized and the RNA partially digested, allowing a small fragment to remain attached to protein. RNA-protein complexes of interest are partially purified by immunoprecipitation and noncovalently associated RNA removed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These purified RNA-protein complexes are isolated and treated with proteinase K, which removes protein but leaves intact RNA. This RNA is abundant enough, and competent for, RNA linker ligation, reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and sequencing. Database matching of these short 70- to 100-nt RNA CLIP (crosslinking and immunoprecipitation of RNA-protein complexes) "tags," which mark the native binding sites of RNA binding proteins, potentially allows the entire target repertoire of an RNA binding protein to be determined. |
Keywords: | Animals Mice RNA-Binding Proteins Cross-Linking Reagents Immunoprecipitation Ultraviolet Rays Protein Binding |
Description: | Previous ed.: 1999. |
DOI: | 10.1007/978-1-60327-475-3_6 |
Published version: | http://dx.doi.org/10.1007/978-1-60327-475-3_6 |
Appears in Collections: | Aurora harvest 5 Molecular and Biomedical Science publications |
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