title: Appropriate real-time PCR reference genes for fluoride treatment studies performed in vitro or in vivo.
abstract: OBJECTIVE
  Quantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo.
  
  
  METHODS
  Five candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5mM sodium fluoride (NaF) for 6 or 18 h followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100 ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples.
  
  
  RESULTS
  The most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene.
  
  
  CONCLUSIONS
  This study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ.
authors.affiliation: Department of Mineralized Tissue Biology and Harvard School of Dental Medicine, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA. Electronic address: dfaibish@forsyth.org.
authors.forename: D
authors.initials: D
authors.lastname: Faibish
authors.affiliation: Department of Mineralized Tissue Biology and Harvard School of Dental Medicine, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA. Electronic address: suzuki.94@osu.edu.
authors.forename: M
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Department of Mineralized Tissue Biology and Harvard School of Dental Medicine, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA. Electronic address: bartlett.196@osu.edu.
authors.forename: J D
authors.initials: JD
authors.lastname: Bartlett
mesh_terms: D000818:Animals; D002460:Cell Line; D004658:Enamel Organ / Q000166:cytology / Q000187:drug effects; D009050:Fluorosis, Dental / Q000209:etiology / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling; D008297:Male; D051379:Mice; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D012313:RNA / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012969:Sodium Fluoride / Q000494:pharmacology*; D051742:Transcription Factor CHOP / Q000235:genetics
chemical_list: C497251:Ddit3 protein, rat; C577475:EEF1A1 protein, rat; D020648:Peptide Elongation Factor 1; D051742:Transcription Factor CHOP; D012313:RNA; D012969:Sodium Fluoride
keywords: Fluoride; Fluorosis; Gene expression; Real-time PCR; Reference genes