Profiling RNA polymerase II using the fast chromatin immunoprecipitation method.
The traditional method for determining the transcription rate of a gene, nuclear run-on, is time consuming, laborious, and involves the use of high levels of radio-labeled nucleotides. When combined with measurements of mRNA levels, RNA polymerase II (Pol II) chromatin immunoprecipitation (ChIP) is a simpler alternative to determine the transcription rate of genes. Moreover, this approach provides more information about the transcriptional regulation of a gene than nuclear run-on. The power of the ChIP assay is that it gives a researcher the ability to not only detect a specific protein-DNA interaction in vivo, for instance with Pol II, but also to determine the relative density of factors along genes or the entire genome. Though powerful, the conventional ChIP assay is time consuming (involving 2 days or more) and involves labor intensive steps. With Fast ChIP we simplified the assay to greatly reduce the time and labor involved. The improved assay is especially useful for studies which involve many samples, including the probing of multiple transcriptionally related factors simultaneously and/or looking at transcription events over several time points. Using Fast ChIP, 24 sheared chromatin samples can be processed to yield PCR ready DNA in 5 h.
Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA. imjoel@u.washington.edu
Joel
J
Nelson
Oleg
O
Denisenko
Karol
K
Bomsztyk
D047369:Chromatin Immunoprecipitation / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
D012319:RNA Polymerase II