Published online 24 June 2004
Nucleic Acids Research, Vol. 32 No. 11 © Oxford University Press 2004; all rights reserved
RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription
Department of Biochemistry and Molecular Biology and 1 Department of Physiology, University of Valencia, 46010 Spain
* To whom correspondence should be addressed. Tel: +349 6354 4867; Fax: +349 6354 4635; Email: gerardo.lopez{at}uv.es
Present addresses: Gaetano Serviddio, Dipartimento di Scienze Mediche e del Lavoro, Università di Foggia, Foggia, Italy
Abedelhalim Boukaba, Wellcome Trust Centre for Cell Biology, ICMB. 6.33 Swann Building, The University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Received April 7, 2004; Revised and Accepted June 6, 2004
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.
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