Identification of target mRNAs of regulatory RNA-binding proteins using mRNP immunopurification and microarrays.
RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To identify novel target mRNAs of RBPs, we incubate total RNA with recombinant RBP and immunoselect the messenger-ribonucleoproteins using a specific anti-RBP antibody. The mRNA composition of the supernatant and/or immunoprecipitated fraction is analyzed using dual-color microarrays in comparison with control reaction. From start to finish, the protocol takes approximately 6 d.
Sanchez
Mayka
M
Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 153, 69120 Heidelberg, Germany.
Galy
Bruno
B
Hentze
Matthias W
MW
Muckenthaler
Martina U
MU
D018076:DNA, Complementary / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; C026452:messenger ribonucleoprotein; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases