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(462i) Microfluidic Native Chromatin Immunoprecipitation (µNChIP) Assay for Histone Acetylation Detection

Geng, T., Purdue University
Bao, N., Purdue University
Litt, M., Indiana University School of Medicine - Muncie at Ball State University
Lu, C., Virginia Tech

Epigenetic modifications, such as acetylation, of the core histone proteins play a role in the control of eukarytotic transcription. To explore the mechanisms, chromatin immunoprecipitation (ChIP) assay is widely applied to studying the presence of various histone modifications at defined genomic regions within living cells or tissues. Native ChIP (NChIP), which uses native chromatin fragments prepared by micrococcal nuclease digestion in the absence of cross-linking, is particularly suitable for detecting histone modifications due to the high affinity between histones and DNA. However, current ChIP assays require a large amount of cells (ideally > 107), and involve lengthy and laborious steps (4 days). Here, we developed an integrated microfluidic chip to permit sensitive and rapid NChIP assay based on a small amount of cells. The characteristic of miniaturization allows a low number of cells to be processed on a microfluidic chip while keeping sufficiently high concentrations of chromatin fragments in microchannel. In addition, microscale magnetic beads (ProActive® Protein A Coated Microspheres, 6.2 μm) with effective reaction surface are used to generate active mixing in order to accelerate the rate of antibody binding onto the target protein, thereby shortening the immunoprecipitation incubation time to 30 min. We assayed histone H3 acetylation at forlate receptor gene of chicken β-globin locus in 6C2 cells using real-time PCR assay. The μNChIP assay including DNA purification and real-time PCR could be completed in 1 day starting from less than 1000 cells. Our microfluidics-based strategy has the potential to be employed for efficient epigenetic analysis of rare samples such as stem cells, embryonic cells and primary tumors.