title: RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Valencia, 46010 Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Sandoval
authors.forename: José L
authors.initials: JL
authors.lastname: Rodríguez
authors.forename: Gema
authors.initials: G
authors.lastname: Tur
authors.forename: Gaetano
authors.initials: G
authors.lastname: Serviddio
authors.forename: Javier
authors.initials: J
authors.lastname: Pereda
authors.forename: Abdelhalim
authors.initials: A
authors.lastname: Boukaba
authors.forename: Juan
authors.initials: J
authors.lastname: Sastre
authors.forename: Luis
authors.initials: L
authors.lastname: Torres
authors.forename: Luis
authors.initials: L
authors.lastname: Franco
authors.forename: Gerardo
authors.initials: G
authors.lastname: López-Rodas
mesh_terms: 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
chemical_list: D002843:Chromatin; D012333:RNA, Messenger; D012319:RNA Polymerase II