title: Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR.
abstract: The "quantitative" ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.
authors: Cardiovascular Division, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia, United States of America. pamela.schoppee.bortz@virginia.edu
authors: Pamela D
authors: PD
authors: Schoppee Bortz
authors: Brian R
authors: BR
authors: Wamhoff
mesh_terms: D047369:Chromatin Immunoprecipitation / Q000379:methods* / Q000592:standards; D019411:Clinical Laboratory Techniques; D004247:DNA / Q000032:analysis*; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods; D013010:Sonication
chemical_list: D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D004247:DNA