Evaluation of candidate RT-qPCR reference genes in the aging African turquoise killifish brain

Reference genes (RGs) are typically used to normalize gene expression from RT-qPCR experiments. However, the expression of commonly used reference genes can vary across different physiological conditions, such as aging, and potentially lead to inaccurate interpretations of results. In African turquoise killifish (N. furzeri), the stability of reference genes has not been evaluated during aging. Here, we evaluate six candidate reference genes used in other models of aging (ActB, Cyc1, Gapdh, Gusb, Oaz1a, and Tbp) and examine their brain expression stability in adult male and females from young (10 weeks post-hatching) to old (25 weeks post hatching). To examine reference gene stability, we used a combination of summary statistics based on analyses of Cq values, normalized fold change of tyrosine hydroxylase (TH), and available computational programs. Overall, we found that Cyc1, Oaz1a, and Gusb were the most stable reference genes during aging, with specific rankings reflecting sex-dependent differences. Gapdh and ActB were the least stable between both sexes across young and old brains. When TH expression was normalized to our selected RGs, we found that only female samples had an age-related decrease in expression, and expression analysis was highly dependent on the choice of reference gene.