RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription.
We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to those of other procedures. However, in genes whose mRNA is more stable (e.g. the hemopexin, hpx, gene) RNAPol-ChIP informs on real-time transcription with results comparable to those of methods such as nuclear run-on or run-off, which require the isolation of highly purified nuclei. Moreover, RNAPol-ChIP advantageously compares with methods based on the analysis of steady-state mRNA (northern blot or RT-PCR). Additional advantages of RNAPol-ChIP, such as the possibility of combining it with classical ChIP analysis to study transcription-associated changes in chromatin are discussed.
Sandoval
Juan
J
Department of Biochemistry and Molecular Biology, University of Valencia, 46010 Spain.
Rodríguez
José L
JL
Tur
Gema
G
Serviddio
Gaetano
G
Pereda
Javier
J
Boukaba
Abdelhalim
A
Sastre
Juan
J
Torres
Luis
L
Franco
Luis
L
López-Rodas
Gerardo
G
D000208:Acute Disease; D000818:Animals; D002843:Chromatin / Q000235:genetics* / Q000302:isolation & purification; D016762:Genes, fos; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008115:Liver Regeneration / Q000235:genetics; D008297:Male; D010195:Pancreatitis / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012319:RNA Polymerase II / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014158:Transcription, Genetic
D002843:Chromatin; D012333:RNA, Messenger; D012319:RNA Polymerase II