Optimal Reference Genes for RT-qPCR Experiments in Hippocampus and Cortex of Rats Chronically Exposed to Excessive Fluoride.

Normalization of the quantitative real-time PCR (RT-qPCR) data to the stably expressed reference genes is critically important for obtaining reliable results. However, all previous studies focused on F toxicity for brain tissues used a single, non-validated reference gene, what might be a cause of contradictory or false results. The present study was designed to analyze the expression of a series of reference genes to select optimal ones for RT-qPCR analysis in cortex and hippocampus of rats chronically exposed to excessive fluoride (F) amounts. Six-week-old male Wistar rats randomly assigned to four groups consumed regular tap water with 0.4 (control), 5, 20, and 50 ppm F (NaF) for 12 months. The expression of six genes (Gapdh, Pgk1, Eef1a1, Ppia, Tbp, Helz) was compared by RT-qPCR in brain tissues from control and F-exposed animals. The stability of candidate reference genes was evaluated by coefficient of variation (CV) analysis and RefFinder online program summarizing the results of four well-acknowledged statistical methods (Delta-Ct, BestKeeper, NormFinder, and GeNorm). In spite of some discrepancies in gene ranking between these algorisms, Pgk1, Eef1a1, and Ppia were found to be most valid in cortex, while Ppia, Eef1a1, and Helz showed the greatest expression stability in hippocampus. Tbp and Helz were identified as the least stable genes in cortex, whereas Gapdh and Tbp are unsuitable for hippocampus. These data indicate that reliable mRNA quantification in the cortex and hippocampus of F-poisoned rats is possible using normalization to geometric mean of Pgk1+Eef1a1 or Ppia+Eef1a1 expression, respectively.