title: Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control.
abstract: BACKGROUND
  Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted.
  
  
  RESULTS
  We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types.
  
  
  CONCLUSIONS
  The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institute and Center for Innovative Medicine, Huddinge, Sweden. shintaro.katayama@ki.se.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Katayama
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institute and Center for Innovative Medicine, Huddinge, Sweden. tiina.skoog@ki.se.
authors.forename: Tiina
authors.initials: T
authors.lastname: Skoog
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institute and Center for Innovative Medicine, Huddinge, Sweden. eeva-mari.jouhilahti@ki.se.
authors.forename: Eeva-Mari
authors.initials: EM
authors.lastname: Jouhilahti
authors.affiliation: Folkhälsan Institute of Genetics, Helsinki, Finland. annika.siitonen@gmail.com.|Department of Medical Genetics, Haartman Institute and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland. annika.siitonen@gmail.com.
authors.forename: H Annika
authors.initials: HA
authors.lastname: Siitonen
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Helsinki, Helsinki, Finland. kristo.nuutila@helsinki.fi.
authors.forename: Kristo
authors.initials: K
authors.lastname: Nuutila
authors.affiliation: Folkhälsan Institute of Genetics, Helsinki, Finland. mari.h.tervaniemi@helsinki.fi.|Department of Medical Genetics, Haartman Institute and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland. mari.h.tervaniemi@helsinki.fi.
authors.forename: Mari H
authors.initials: MH
authors.lastname: Tervaniemi
authors.affiliation: Helsinki Burn Center, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland. jyrki.vuola@hus.fi.
authors.forename: Jyrki
authors.initials: J
authors.lastname: Vuola
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. anna.johnsson@ki.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Johnsson
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. peter.lonnerberg@ki.se.
authors.forename: Peter
authors.initials: P
authors.lastname: Lönnerberg
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. sten.linnarsson@ki.se.
authors.forename: Sten
authors.initials: S
authors.lastname: Linnarsson
authors.affiliation: Folkhälsan Institute of Genetics, Helsinki, Finland. outi.elomaa@helsinki.fi.|Department of Medical Genetics, Haartman Institute and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland. outi.elomaa@helsinki.fi.
authors.forename: Outi
authors.initials: O
authors.lastname: Elomaa
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Helsinki, Helsinki, Finland. esko.kankuri@helsinki.fi.
authors.forename: Esko
authors.initials: E
authors.lastname: Kankuri
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institute and Center for Innovative Medicine, Huddinge, Sweden. juha.kere@ki.se.|Department of Medical Genetics, Haartman Institute and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland. juha.kere@ki.se.|Science for Life Laboratory, Solna, Sweden. juha.kere@ki.se.
authors.forename: Juha
authors.initials: J
authors.lastname: Kere
mesh_terms: D017209:Apoptosis / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D004817:Epidermis / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D012313:RNA / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017423:Sequence Analysis, RNA; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D016038:Skin Transplantation
chemical_list: D012333:RNA, Messenger; D012313:RNA