title: Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.
abstract: There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.
authors.affiliation: Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Harrachgasse 21/7, 8010, Graz, Austria. thomas.kroneis@medunigraz.at.|Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden. thomas.kroneis@medunigraz.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kroneis
authors.affiliation: Diabetes and Metabolism Unit, School of Clinical Sciences, Southmead Hospital, University of Bristol, Bristol, UK.
authors.forename: Jody
authors.initials: J
authors.lastname: Ye
authors.affiliation: Diabetes and Metabolism Unit, School of Clinical Sciences, Southmead Hospital, University of Bristol, Bristol, UK.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Gillespie
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics*; D005629:Frozen Sections; D005838:Genotype; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods*; D010179:Pancreas / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D004247:DNA
keywords: Diabetes; Laser microdissection and pressure catapulting; Pancreatic cells; Tissue