Ribonucleoprotein immunoprecipitation (RNP-IP): a direct in vivo analysis of microRNA-targets.
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.
Hassan
Mohammad Q
MQ
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Gordon
Jonathan A R
JA
Lian
Jane B
JB
van Wijnen
Andre J
AJ
Stein
Janet L
JL
Stein
Gary S
GS
D020413:3' Untranslated Regions; D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D047468:Immunoprecipitation; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*
D020413:3' Untranslated Regions; D017931:DNA Primers; D035683:MicroRNAs; D012261:Ribonucleoproteins