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 RGs can vary across different physiological conditions, such as aging, and potentially lead to inaccurate interpretations of results. In African turquoise killifish (Nothobranchius 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 males and females from young (10 weeks post-hatching) to old (25 weeks post-hatching). To examine RG 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 across both sexes, with specific rankings reflecting sex-dependent differences, while gapdh and actb were the least reliable. Importantly, 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. Taken together, our findings provide the first systematic evaluation of RG stability in the killifish brain and highlight cyc1, oaz1a, and gusb as reliable RGs for studies of aging. We recommend that future studies use at least two of these RGs in combination for accurate normalization and evaluate RGs for selected experimental conditions within the framework established in this study.