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

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.

SUBMITTER: Dahl JA

PROVIDER: S-EPMC2241883 | biostudies-other | 2008 Feb

REPOSITORIES: biostudies-other

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

Dahl John Arne JA Collas Philippe P

Nucleic acids research 20080117 3

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 transcrip ...[more]

PMID: 18202078

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Project description:Determination of tumour-specific transcription based on liquid biopsies possesses a large diagnostic and prognostic potential in non-small cell lung cancer (NSCLC). Cell-free DNA (cfDNA) packed in nucleosomes mirrors the histone modification profiles present in the cells of origin. H3 lysine 36 trimethylation (H3K36me3)-modified nucleosomes are associated with active genes, and therefore, cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-associated cfDNA has the potential to delineate whether transcription of a particular gene is occurring in the cells from which its cfDNA originates. We hypothesized that cfChIP can delineate transcriptional status of genes harbouring somatic cancer mutations and analysed the recurrently observed EGFR-L858R mutation as an example. In representative NSCLC cell lines, the relationship between wild-type (WT) and mutated EGFR transcriptional activity and mRNA expression levels was analysed using H3K36me3 ChIP and EGFR mRNA reverse transcription quantitative PCR (RT-qPCR), respectively. The ChIP analysis showed that both WT and mutated EGFR are transcribed and that mRNA is similarly expressed per EGFR copy. Based on this observation, we proceeded with EGFR cfChIP using blood plasma from NSCLC patients harbouring the EGFR-L858R mutation. EGFR-WT fragments can originate from both nontumour cells with no or low EGFR transcription and tumour cells with active EGFR transcription, whereas EGFR-L858R fragments must specifically originate from tumour cells. H3K36me3 cfChIP followed by droplet digital PCR (ddPCR) revealed significantly higher enrichment of EGFR-L858R compared to EGFR-WT fragments. This is in alignment with EGFR-L858R being actively transcribed in the NSCLC tumour cells. This study is proof-of-principle that cfChIP can be used to identify tumour-specific transcriptional activity of mutated alleles, which can expand the utility of liquid biopsy-based cfDNA analyses to enhance tumour diagnostics and therapeutics.

Dataset Information

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

Publications

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

Similar Datasets

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