title: Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.
abstract: OBJECTIVE
  Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing.
  
  
  METHODS
  Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs.
  
  
  RESULTS
  Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome.
  
  
  CONCLUSIONS
  RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.
authors.affiliation: a Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty , University of Cologne , Cologne , Germany.|b Center for Biochemistry, Medical Faculty , University of Cologne , Cologne , Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Etich
authors.affiliation: a Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty , University of Cologne , Cologne , Germany.|b Center for Biochemistry, Medical Faculty , University of Cologne , Cologne , Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Bergmeier
authors.affiliation: a Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty , University of Cologne , Cologne , Germany.|b Center for Biochemistry, Medical Faculty , University of Cologne , Cologne , Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Pitzler
authors.affiliation: a Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty , University of Cologne , Cologne , Germany.|b Center for Biochemistry, Medical Faculty , University of Cologne , Cologne , Germany.
authors.forename: Bent
authors.initials: B
authors.lastname: Brachvogel
mesh_terms: D000818:Animals; D024042:Collagen Type I; D005786:Gene Expression Regulation; D053583:Interleukin-1beta / Q000096:biosynthesis; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D051379:Mice; D035683:MicroRNAs / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D012867:Skin / Q000293:injuries / Q000378:metabolism / Q000473:pathology; D014945:Wound Healing; D014947:Wounds and Injuries / Q000378:metabolism* / Q000473:pathology
chemical_list: D024042:Collagen Type I; C508585:IL1B protein, human; D053583:Interleukin-1beta; D035683:MicroRNAs; D012333:RNA, Messenger; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse
keywords: RNU6B; mRNA; miRNA; reference gene; wound healing