Comparative evaluation of four reference genes for qRT-PCR assays in snow trout (Schizothorax richardsonii) under multiple experimental conditions

The analytical accuracy of gene expression analysis largely depends on the stability of the reference gene(s) used for normalization of target mRNA expression under different experimental conditions. In this study, we characterized three reference genes: beta-actin (β-act), elongation factor 1 alpha (ef1α), and glyceraldehyde 3-phosphate dehydrogenase (gapdh) in the Trans-Himalayan snow trout, Schizothorax richardsonii. Subsequently, we evaluated their stability, along with 18 S rRNA, under eight experimental conditions using the geNorm, NormFinder, BestKeeper, and delta CT algorithms. The results indicated that 18 S rRNA was the most stable reference gene across sixteen tissue types, whereas ef1α showed the highest stability during early developmental stages (0-21 days post-hatch) and between sexes (male and female muscle tissue). In other instances, β-act was the most stable reference gene in snow trout muscle across different age groups (0 + and 2 + years), nutritional conditions (starvation-refeeding and varying dietary protein and lipid levels), and rearing temperatures (6, 12, 18, and 24 °C). In contrast, gapdh exhibited poor stability and was regulated by low temperature, making it unsuitable as a reference gene under such conditions. For further validation, we performed relative quantification of primary myogenic regulators (myod and myf5) at different rearing temperatures using the most and least stable reference gene(s) as normalizers. The results reaffirmed the importance of selecting stable reference gene(s) for qRT-PCR assays, as the expression patterns of the myogenic regulators were significantly incorrect when normalized using the least stable reference genes. To our knowledge, this is the first comprehensive evaluation of internal reference genes for gene expression analysis in a coldwater cyprinid across different tissues, developmental stages, age, sex, nutritional conditions and temperature regimes.