Identifying stable reference genes for evaluation of antioxidative enzyme gene expression in auditory cortex and cochlea of young and old Fischer 344 rats

Abstract

Conclusion. This study suggests that the validation of reference genes is important for the accurate evaluation of differential gene expression in age-related hearing loss (AHL). Objectives. To identify the most stable reference gene in the auditory cortex and cochlea of young and old rats. Materials and methods. mRNA levels of some common reference genes such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C, hypoxanthine phosphoribosyl-transferase and eukaryotic translation elongation factor (EF) were quantified in the auditory cortex and cochlea of young and old rats by quantitative RT-PCR, and then their stability was evaluated using GeNorm and Normfinder software. Results. There were some variations in the expression of reference genes found in the auditory cortex and cochlea of rats, wherein EF and GAPDH were identified as the most stable reference genes in auditory cortex and cochlea, respectively. mRNA levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase and glutathione peroxidase in the auditory cortex and cochlea do not vary significantly in young and old rats, when they are normalized with EF or GAPDH, the valid reference genes, respectively. However, Cu/Zn-SOD and catalase levels differ significantly in the auditory cortex when they are normalized with β-actin, an invalid reference gene, resulting in the misinterpretation of the target gene expression levels.