Ribonucleoprotein immunoprecipitation (RNP-IP): a direct in vivo analysis of microRNA-targets

Abstract

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.