Validation of Reference Genes for RT–qPCR Analysis in Noise–Induced Hearing Loss: A Study in Wistar Rat

The reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT–qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT–qPCR has been widely used to elucidate molecular correlates of noise–induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b–Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT–qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise–exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT–qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top–ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT–qPCR analysis in studies of NIHL using Wistar rats as animal model.