Appropriate real-time PCR reference genes for fluoride treatment studies performed in vitro or in vivo

• Identification of reference genes for fluoride qPCR experiments is proposed.
• RNA was isolated from LS8 ameloblast-derived cells and rat enamel organs.
• qPCR experiments must first validate the stability of reference gene expression in context of the specified conditions.
• Gapdh and Eef1a1 were the most stably expressed genes in enamel organs from fluoride-treated rats.

ObjectiveQuantitative 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.DesignFive 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 5 mM 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.ResultsThe 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.ConclusionsThis 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.