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. 2008 Feb;36(3):e15.
doi: 10.1093/nar/gkm1158. Epub 2008 Jan 17.

MicroChIP--a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies

John Arne Dahl  1 , Philippe Collas
Affiliations

MicroChIP--a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies

John Arne Dahl et al. Nucleic Acids Res. 2008 Feb.

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein-DNA interactions. Drawbacks of current ChIP assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy procedures with limited applications. There are to date no protocols for fast and parallel ChIPs of post-translationally modified histones from small cell numbers or biopsies, and importantly, no protocol allowing for investigations of transcription factor binding in small cell numbers. We report here the development of a micro (micro) ChIP assay suitable for up to nine parallel quantitative ChIPs of modified histones or RNA polymerase II from a single batch of 1000 cells. MicroChIP can also be downscaled to monitor the association of one protein with multiple genomic sites in as few as 100 cells. MicroChIP is applicable to small fresh tissue biopsies, and a cross-link-while-thawing procedure makes the assay suitable for frozen biopsies. Using MicroChIP, we characterize transcriptionally permissive and repressive histone H3 modifications on developmentally regulated promoters in human embryonal carcinoma cells and in osteosarcoma biopsies. muChIP creates possibilities for multiple parallel and rapid transcription factor binding and epigenetic analyses of rare cell and tissue samples.

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Figures

Figure 1.
Figure 1.
Development and validation of μChIP. (A) Genomic regions examined. Bars and numbers indicate the position of ChIP amplicons relative to the transcription start site (TSS). PE, PP and CR1 delineate the proximal enhancer, proximal promoter and conserved region 1 of OCT4, respectively. (B) RT-PCR analysis of expression of GAPDH, OCT4, NANOG and SLC10A6 in NCCIT cells. Plus (+) and minus (−) indicate the presence or absence of reverse transcriptase (RT). (C–E) ChIP analysis of H3 modifications (x-axis) in NCCIT cells on promoters of indicated genes. (C) Analysis from chromatin of 100 000 cells, (D) 1000 cells and (E) 100 cells. (F–H) ChIP analysis of RNAPII binding to GAPDH, OCT4 and SLC10A6 promoters in the same chromatin samples as those examined for histones in (C–E). In (C–H), data are expressed as percent precipitation relative to input chromatin (mean ± SD; 2–9 independent experiments).
Figure 2.
Figure 2.
μChIP is suitable for analysis of histone or RNAPII binding in as few as 100 cells. (A) ChIP analysis of H3K9ac, H3K4m3 and H3K9m3 in separate 100-cell samples for each antibody, and for a no-antibody (No Ab) control. (B) ChIP analysis of RNAPII association with the GAPDH, NANOG, OCT4 and SLC10A6 promoters 100-cell samples. Two to four ChIPs were performed with each antibody. Data are expressed as in Figure 1.
Figure 3.
Figure 3.
μChIP analysis of the association of modified histones and RNAPII to GAPDH, OCT4, NANOG and SLC10A6 promoters in human osteosarcoma biopsies. (A) ChIP analysis of fresh and frozen/thawed tissue samples (data from duplicate ChIPs from each sample). (B) RT-PCR analysis of expression of the respective genes (30 PCR cycles). Plus (+) and minus (−) indicate the presence or absence of reverse transcriptase (RT).
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