title: Fetal gut laser microdissection in combination with RNA preamplification enables epithelial-specific transcriptional profiling.
abstract: Laser microdissection (LMD) technology enables highly specific gene expression analyses of biologically relevant questions at cell- or tissue-specific resolution. Nevertheless, specific cell types are often limited in quantity (i.e. fetal tissue), making high quality RNA extraction and subsequent gene expression approaches via common reverse transcriptase-quantitative PCR (RT-q-PCR) challenging. In the case of fetal gut epithelia representing immune modulatory interphases gene expression analysis with common RT-q-PCR is limited to a few genes (<10). To circumvent these limitations we provide a workflow using laser microdissection of 1.5Mioμm(2) dissected area of murine fetal intestinal epithelial cells (IEC) from fetal ileum and colon with subsequent RNA isolation, whole transcriptome preamplification (WTA) and gene expression analysis by microarray and quantitative PCR (qPCR). This workflow allows simultaneous analyses of global (microarrays) and targeted gene expression (qPCR) and consequently increases the number of measurable genes up to 25-fold by qPCR. It is suitable for cryosections from many tissues and species in order to evaluate in utero biological effects on specific effector sites.
authors.affiliation: Chair of Nutrition and Immunology, Technische Universität München, Freising-Weihenstephan, Germany; ZIEL - Research Center for Nutrition and Food Sciences, Freising-Weihenstephan, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Hemmerling
authors.affiliation: Wageningen University, Nutrition, Metabolism and Genomics Group, Bomenweg 2, NL-6703 HD Wageningen, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Jansen
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: M
authors.initials: M
authors.lastname: Müller
authors.affiliation: Chair of Nutrition and Immunology, Technische Universität München, Freising-Weihenstephan, Germany; ZIEL - Research Center for Nutrition and Food Sciences, Freising-Weihenstephan, Germany. Electronic address: Dirk.Haller@tum.de.
authors.forename: D
authors.initials: D
authors.lastname: Haller
mesh_terms: D000818:Animals; D004847:Epithelial Cells / Q000378:metabolism*; D005333:Fetus / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods; D007413:Intestinal Mucosa / Q000378:metabolism*; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D012313:RNA
keywords: Fetal intestinal epithelium; Laser microdissection; Transcriptional profiling; Whole transcriptome preamplification