Abstract
BackgroundThe recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required.ResultsIn this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-rasv12 transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II).ConclusionsEpigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.
Highlights
The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples
Application of PAT-ChIP to the study of core needle biopsies We first evaluated the application of PAT-ChIP to Core needle biopsy (CNB) obtained from FFPE samples derived from a murine model of acute promyelocytic leukemia (APL)
The spleen CNBs (0.6 mm in diameter) were fragmented by sonication (15 pulses of 15 s at 85% of amplitude): probably due to a lower specific surface than a FFPE section of equal tissue volume, a CNB needs to be sonicated by applying a higher total energy than that normally used for a 10-μm-thick FFPE tissue section
Summary
The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. To take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required. Total DNA obtained by ChIP is mainly analyzed at the level of single sequences by quantitative PCR (qPCR; locus-specific studies), or ‘genome-wide’ by ChIP-Seq, in order to investigate the distribution of the protein of interest over the entire genome [11,12]. These studies are producing an enormous amount of complex information that is strongly contributing to the elucidation of the epigenetic alterations involved in tumor development. Many authors believe that the time when epigenetic biomarkers (prognostic or even predictive) and/or specific epigenetic targets will start to be used in clinical practice is not far away [13,14]
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