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Ribonucleoprotein immunoprecipitation (RNP‐IP): A direct in vivo analysis of microRNA‐targets
Author(s) -
Hassan Mohammad Q.,
Gordon Jonathan A.R.,
Lian Jane B.,
van Wijnen Andre J.,
Stein Janet L.,
Stein Gary S.
Publication year - 2010
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.22562
Subject(s) - immunoprecipitation , ribonucleoprotein , microrna , untranslated region , biology , microbiology and biotechnology , messenger rna , rna binding protein , rna , gene , genetics
We have developed a novel Ribonucleoprotein Immunoprecipitation (RNP‐IP) method to isolate miR‐RISC complexes, associated microRNAs and target mRNAs. Our method characterizes mRNAs present in immunoprecipitates containing miR‐RISC complexes that were obtained using GW182 and AGO2 antibodies. MicroRNA bound transcripts were reverse transcribed and amplified using seed sequence and 3′UTR derived primers. This flexible IP‐based assay is a straightforward method to identify miRs participating in gene regulation and their cognate mRNAs in real time. J. Cell. Biochem. 110: 817–822, 2010. © 2010 Wiley‐Liss, Inc.