title: PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  In 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-ras(v12) 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).
  
  
  CONCLUSIONS
  Epigenetic 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.
authors.affiliation: Department of Biomolecular Sciences, University of Urbino 'Carlo Bo', Molecular Pathology Lab. 'PaoLa', Via Arco d'Augusto, 2, Fano 61032, Italy ; Department of Experimental Oncology, European Institute of Oncology, Via Adamello, 16, Milan 20139, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Amatori
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello, 16, Milan 20139, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Ballarini
authors.affiliation: Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via Francesco Sforza, 33, Milan 20122, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Faversani
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello, 16, Milan 20139, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Belloni
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello, 16, Milan 20139, Italy.
authors.forename: Fulvia
authors.initials: F
authors.lastname: Fusar
authors.affiliation: Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via Francesco Sforza, 33, Milan 20122, Italy ; Department of Pathophysiology and Transplantation, University of Milan, Via Francesco Sforza, 35, Milan 20122, Italy.
authors.forename: Silvano
authors.initials: S
authors.lastname: Bosari
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello, 16, Milan 20139, Italy.
authors.forename: Pier Giuseppe
authors.initials: PG
authors.lastname: Pelicci
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Via Adamello, 16, Milan 20139, Italy ; Department of Biosciences, University of Milan, Via Giovanni Celoria, 26, Milan 20133, Italy.
authors.forename: Saverio
authors.initials: S
authors.lastname: Minucci
authors.affiliation: Department of Biomolecular Sciences, University of Urbino 'Carlo Bo', Molecular Pathology Lab. 'PaoLa', Via Arco d'Augusto, 2, Fano 61032, Italy.
authors.forename: Mirco
authors.initials: M
authors.lastname: Fanelli
keywords: Chromatin immunoprecipitation; FFPE samples; Laser microdissection; PAT-ChIP; Pathology samples
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