title: The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation.
abstract: Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.
authors.affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: A B
authors.initials: AB
authors.lastname: Eberle
authors.forename: S
authors.initials: S
authors.lastname: Böhm
authors.forename: A-K
authors.initials: AK
authors.lastname: Östlund Farrants
authors.forename: N
authors.initials: N
authors.lastname: Visa
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D002843:Chromatin / Q000276:immunology / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000592:standards; D004247:DNA / Q000737:chemistry*; D004076:Digoxigenin / Q000737:chemistry; D004330:Drosophila / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000276:immunology / Q000378:metabolism; D006801:Humans; D012015:Reference Standards
chemical_list: D000906:Antibodies; D002843:Chromatin; D006657:Histones; D004247:DNA; D004076:Digoxigenin