Abstract B58: Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control

Abstract

Abstract Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate interaction of proteins with specific genomic DNA regions in vivo to provide a better understanding of mechanisms of gene regulation, DNA replication and DNA repair. However, variations in the efficiency of immunoprecipitation, background signals from carryover in immunoprecipitation, variability between chromatin preps, and loss of material during immunoprecipitation and purification of the ChIP DNA are sources of variability that restrict the use of ChIP as a quantitative tool. To address these limitations, we have developed a simple spike-in control that enables labs to generate results that are more consistent and more quantitative. The approach we have developed uses a synthetic peptide-DNA reagent that is spiked into chromatin immunoprecipitation reactions. Because this reagent mimics the behavior of cross-linked chromatin and can be quantitatively recovered by immunoprecipitation, it can be readily incorporated in to virtually any ChIP protocol. To utilize this internal control, a fixed amount of the synthetic peptide-DNA complex is spiked into the chromatin and co-ChIPed simultaneously with the antibody of interest. The recovered synthetic DNA is then used to normalize the results obtained with the antibody of interest. This spike-in control can be designed using a peptide specific to the ChIP antibody of interest and recovered using a single antibody. Alternatively, a universal spike-in control can be designed using a peptide that is recognized by an epitope tag antibody. This universal spike-in control and epitope tagged antibody are used along with the antibody of interest in a standard ChIP reaction. Our data show that the inclusion of our internal control significantly reduces variability between ChIP assays, thus increasing the accuracy of the ChIP data. Because of the universal feature, this method can also be used to compare ChIP data between different antibodies or to normalize data variations that often result between users and multiple experiments. Furthermore, experiments are underway to confirm that such a spike in control can be incorporated upstream of chromatin preparation, and is capable of being adapted for NGS library construction to aid in normalization between chromatin NGS experiments. Citation Format: John M. Rosenfeld, Zirong Li, Konstantin Taganov, Tracy Cooke, Bhaskar Thyagarajan, Alejandra Solache. Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B58.