title: Identification of suitable reference genes in the mouse placenta.
abstract: BACKGROUND
  Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a reliable tool to analyse gene expression profiles. The expression of housekeeping genes generally serves as a reference for mRNA amount, assuming that it remains stable under pathophysiological and experimental conditions. To date, an empirical validation of reference genes suitable for RT-qPCR-based studies in the mouse placenta is missing.
  
  
  METHODS
  We used NormFinder and BestKeeper statistical software to analyse the expression stability of candidate housekeeping genes quantified by RT-qPCR in mouse placentas.
  
  
  RESULTS
  Fifteen of 32 potential candidate housekeeping genes analysed on gestation day (gd) 16.5 in mouse placentas exhibited an optimal cycle threshold (Ct). Among them B2m, Polr2a, Ubc, and Ywhaz genes showed the highest expression stability in placentas from control, but also experimentally-challenged mice. These genes as well as the currently widely used housekeeping genes Hprt1, Actb, and Gapdh were selected for further quality assessments. We quantified the Ct values of these selected genes in placental samples obtained from wild-type or genetically engineered dams at different gds, or upon selected experimental interventions known to affect placental phenotype. Among all housekeeping genes analysed, Polr2a was the most stably expressed and its expression stability excelled in combination with Ubc.
  
  
  CONCLUSIONS
  Polr2a, especially in combination with Ubc, can be proposed as highly suitable endogenous reference for gene expression analysis in mouse-derived placental tissue. Moreover, the validation of both genes as a stable reference gene in human placenta-derived tissue strengthens the translational relevance of RT-qPCR findings using mouse placenta.
authors.affiliation: Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address: e.solano@uke.de.
authors.forename: María Emilia
authors.initials: ME
authors.lastname: Solano
authors.affiliation: Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Thiele
authors.affiliation: Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Mirka Katharina
authors.initials: MK
authors.lastname: Kowal
authors.affiliation: Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Prenatal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Petra Clara
authors.initials: PC
authors.lastname: Arck
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D020043:Genes, Essential; D005819:Genetic Markers; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010920:Placenta / Q000737:chemistry / Q000378:metabolism*; D011247:Pregnancy; D012319:RNA Polymerase II / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards
chemical_list: D000951:Antigens, Neoplasm; D005819:Genetic Markers; D012333:RNA, Messenger; C440529:UBC antigen; D012319:RNA Polymerase II
keywords: Endogenous control; Mouse placenta; Quantitative real-time reverse transcription polymerase chain reaction; Reference gene