Selection of Reference Gene for Expression Studies in the Ovary and Pituitary of Spotted Scat (Scatophagus argus) at Different Ovarian Stages

The spotted scat (Scatophagus argus) is the most widely commercially cultured fish in eastern and southern Asia that possess potential economic value. However, up to date, there are no studies reported on the selection of suitable reference genes in this fish, which is crucial for guaranteeing accurate and reliable results in RT-qPCR analyses. This study aimed at screening the most stable reference genes in the ovary and pituitary at different ovarian stages, which is of great significance for further research on the reproductive regulation mechanism of female S. argus. In this study, we selected twenty-one genes as the candidate reference genes, including four genes in the ovary (EEF1A, ACTB, B2M, and HPRT1), and seventeen genes in the pituitary (B2M, APOA1, CGBA, RPLP0, GNRHR, GUSB, TBP, SLC25A5, RPL4, PLA1A, GAPDH, GNB2L1, CTFS, HPRT1, RPS2, TFRC, and TUBB4B). To analyze the stability of the potential reference genes, we first used four commonly used software, BestKeeper, GeNorm, NormFinder, and Delta Ct, and then we established comprehensive rankings with ReFinder. The results indicate that RPL4 and GNB2L1, and EEF1A and HPRT1 were the most appropriate reference genes for the pituitary and ovary of S. argus, respectively, in the three developmental stages of the ovary. Additionally, GeNorm analysis suggested that for the accurate normalization of gene expression, two reference genes were reliable in the ovary and pituitary of S. argus. To the best of our knowledge and understanding, this is the first study to identify appropriate reference genes for analyzing gene expression in S. argus. Consequently, this study can be used as a foundation for future research on the reproduction of S. argus.