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The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2012 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 426, no 2, 147-152 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2012. Vol. 426, no 2, 147-152 p.
Keyword [en]
ChIP, Quantitative analysis, Normalization, Digoxigenin
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-80438DOI: 10.1016/j.ab.2012.04.020ISI: 000305546000011OAI: oai:DiVA.org:su-80438DiVA: diva2:555363
Note

AuthorCount:4;

Available from: 2012-09-19 Created: 2012-09-19 Last updated: 2017-12-07Bibliographically approved

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Eberle, Andrea B.Böhm, StefanieFarrants, Ann-Kristin ÖstlundVisa, Neus
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