Nucleic Acids Research Advance Access published online on October 21, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn735
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Methods Online |
Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies
1Division of Cancer Genomics and Proteomics, 2Division of Signaling Biology, Ontario Cancer Institute, University Health Network, 3Department of Medical Biophysics, University of Toronto, Toronto, ON, M5G 2M9 and 4Department of Computer Science, University of Toronto, Toronto, ON, M5S 1A8, Canada
*To whom correspondence should be addressed. Tel: +416 946 2276; Fax: +416 946 2840; Email: lpenn{at}uhnres.utoronto.ca
Received August 23, 2008. Revised September 30, 2008. Accepted October 1, 2008.
High-throughput, microarray-based chromatin immunoprecipitation (ChIP-chip) technology allows in vivo elucidation of transcriptional networks. However this complex is not yet readily accessible, in part because its many parameters have not been systematically evaluated and optimized. We address this gap by systematically assessing experimental-design parameters including antibody purity, dye-bias, array-batch, inter-day hybridization bias, amplification method and choice of hybridization control. The combined performance of these optimized parameters shows a 90% validation rate in ChIP-chip analysis of Myc genomic binding in HL60 cells using two different microarray platforms. Increased sensitivity and decreased noise in ChIP-chip assays will enable wider use of this methodology to accurately and affordably elucidate transcriptional networks.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.