Cross-platform detection of epigenetic modifications from extracted chromatin in leucocytes from blood

Abstract

Chromatin contains valuable epigenetic information comprising of both DNA and post translational histone modifications. Traditionally, detection of epigenetic modifications on DNA and histone proteins requires different and elaborate preparation steps. In this study we report on a facile and unique approach for the simultaneous detection and quantification of epigenetic modifications on DNA and histone proteins in one sample preparation step. Our novel one-pot technique consists of a chromatin extraction step from whole blood using bi-functional carboxyl-functionalized magnetic nanoparticles as solid-phase absorbents. Leucocytes enrichment from blood and chromatin extracted from the leucocytes by lysis was achieved with the same carboxyl-functionalized magnetic nanoparticles. The isolated chromatin was then eluted from the magnetic nanoparticles with PBS 1×. Using the eluted chromatin as substrate, we developed a quantitative method for simultaneous detection of epigenetic modifications on DNA and histone proteins by a biotin–streptavidin mediated enzyme-based immunosorbent assay (two-step EIA) on a 96-well plate. The simultaneous detection of epigenetic modifications on DNA and histone was validated by DNA-based EIA and histone-based Western blotting analysis performed separately with conventional protocols. By coupling cell separation and chromatin purification with a simple detection module, the global epigenetic information could be evaluated in less than 8 h in leucocytes from blood. Our simplified cross-platform approach can be used by most laboratories for multiplex detection, uses non-hazardous materials and could be integrated with microfabrication methods for onchip analysis.