Chip‐on‐beads: Flow‐cytometric evaluation of chromatin immunoprecipitation

Abstract

Chromatin immunoprecipitation (ChIP) is a widely used technique for the detection of in vivo DNA-protein interactions underlying epigenetic regulation. The standard readout of ChIP is based on semi-quantitative or quantitative PCR measurements; however, the development of alternative platforms with high throughput potentialities is expected to facilitate the introduction of this method into routine diagnostics. We have established a flow-cytometry-based alternative for the evaluation of ChIP data. The method is based on the capture of the products of a conventional PCR run to low cycle numbers, on microbeads. In vivo histone H4 acetylation and H3 lysine 4 methylation was detected at the promoter of the tissue transglutaminase type 2 gene. These results were confirmed by QPCR measurements. The levels of modifications decreased significantly upon apoptosis and were accompanied by the down-regulation of TGM2 mRNA expression. This method that we named ChIP-on-beads, a combination of flow cytometry and conventional PCR, is a reliable and efficient alternative in the quantitative analysis of ChIP results, especially promising when high throughput monitoring of epigenetic markers of diagnostic importance is required. The method is simple enough to be easily implemented in a routine flow-cytometric laboratory.