Characterization of reference genes in rare minnow, Gobiocypris rarus (Actinopterygii: Cypriniformes: Cyprinidae), in early postembryonic development and in response to EDCs treatment

Background. Endocrine disrupting chemicals (EDCs) are natural and anthropogenic compounds discharged into the environment known to disrupt the endocrine system of humans and animals by mimicking functions of steroids in vivo. Many important events occurring during early postembryonic development, in relation to the gene expression attracted our attention. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible method for gene expression analysis, with gene expression levels quantified by normalization to reference gene. The aim of this study was to select the suitable reference gene after EDCs exposure and during early postembryonic development. Materials and methods. For the study of the fish age effect, juveniles of Gobiocypris rarus Ye et Fu, 1983, were obtained at: 18, 22, 26, 30, 34, 38, 42, 46, and 50 days post fertilization (dpf). For mRNA expression analysis of the juvenile fish after EDCs treatment, the juveniles at 31 dpf were exposed to bisphenol A (BPA) (10 nM) and 17α-ethinylestradiol (EE2) (1 nM), respectively dissolved in dimethyl sulfoxide (DMSO) or solvent (0.001% DMSO, v/v) control group for 3 days. Cq values of the reference genes were obtained using qRT-PCR. The stability of these reference genes was analyzed by BestKeeper, geNorm, and NormFinder software, respectively. The expression of each reference gene was calculated using the 2–ΔCq method. In parallel, the mRNA expressions of cyp19a1b were normalized by the single most/least stable reference gene and the combinations of top-ranked reference genes.  Results. In this study, six candidate reference genes, actb, ef1a, gapdh, g6pd, tbp, and tuba1, were chosen to analyze their expression stability in relation to fish age and in the juvenile fish exposed to BPA and EE2. During early postembryonic development of Gobiocypris rarus, actb，ef1a, and gapdh were identified as the most stably expressed reference genes. In the juvenile fish exposed to BPA and EE2 for three days, gapdh, and actb were the most stable. However, g6pd and tuba1 were identified as the least stably expressed genes during the early postembryonic development and under BPA and EE2 exposure.  Conclusion. The presently reported study suggested that the mRNA expressions of the reference genes could be affected by chemical exposure or different physiological periods. In addition, it was indicated that stable reference gene should be selected to normalize the target gene expression to assure the correctness and accuracy of the experiment results. The last but not the least, we successfully obtained five commonly used reference genes of Gobiocypris rarus Ye et Fu, 1983, which can be applied in future studies serving as the stable reference gene and providing a broader range of selecting the stable reference gene.