Screening and validation of reference genes for qPCR analysis in gonads and embryos of Takifugu bimaculatus

Abstract

Suitable reference genes are one of the necessary conditions for obtaining reliable results by real-time fluorescence quantitative PCR (qPCR). In this study, the expression of the 10 common candidate reference genes (18s rRNA, rps27, cnbp, rpl7, ube2, hsp-at, gapdh, β-actin, rpl13a, 1-ef1a) at different developmental stages of gonad and embryo of Takifugu bimaculatus were analyzed by qPCR. And the expression stability of these reference genes is analyzed by GeNorm, NormFinder and Bestkeeper softwares. The results showed that the expression stability of 1-ef1a was the highest (p < 0.05) combined with the results of three analysis softwares, and the optimal number of reference genes was 3, which were 1-ef1a, hsp-at and rps27. Based on this result, we analyzed the expression of objective genes (foxl2 and dmrt1) at different developmental stages (early, medium-term and late) of gonads and embryos (eye vesicle stage, motility stage, heart anlage stage, retinal pigmentation stage, newly hatched larva) of T. bimaculatus by qPCR. Among which the highest expression level of foxl2 was in the middle stage of ovary (p < 0.05), and dmrt1 was expressed in the early stage of testis with the highest level (p < 0.05). The expression levels of foxl2 and dmrt1 in the early stages of embryonic development were low or even almost non-existent, and had a slight increase in the late embryonic development (newly hatched larva). The expression profile of objective genes are consistent with their roles in the development of gonads and embryos, which indicates that 1-ef1a, hsp-at and rps27 are the suitable reference gene combination for detecting the expression of objective genes by qPCR in gonads and embryos of T. bimaculatus. This research also provides the reliability data for screening the reference genes by qPCR in fish.