Selection of suitable candidate genes for miRNA expression normalization in Yellow River Carp (Cyprinus carpio. var)

Yellow River carp is widely cultivated in the world due to its economic value in aquaculture, and the faster growth of females compared to males. It is believed that microRNAs (miRNA) are involved in gonadal differentiation and development. qPCR is the most preferred method for miRNA functional analysis. Reliable reference genes for normalization in qRT-PCR are the key to ensuring the accuracy of this method. The aim of present research was to evaluate as well as identify the efficacy of reference genes for miRNA expression using qRT-PCR in Yellow River carp. Nine ncRNAs (miR-101, miR-23a, let7a, miR-26a, miR-146a, miR-451, U6, 5S, and 18S) were chosen and tested in four sample sets: (1) different tissues in adult carp, (2) different tissues in juvenile carp, (3) different early developmental stages of carp, and (4) different developmental stages of carp gonads. The stability and suitability values were calculated using NormFinder, geNorm, and BestKeeper software. The results showed that 5S was a suitable reference gene in different tissues of adult and juvenile carp. The genes 5S, 18S, and U6 were the most stable reference genes in the early developmental stages of carp. Let-7a and miR-23a were considered as the suitable reference genes in the development of gonads. All these reference genes were subsequently validated using miR-430. The results showed that genes 5S and 18S were the most suitable reference genes to normalize miRNA expression under normal growth conditions in early different developmental stages. The genes Let-7a, and miR-23a were the most suitable in different developmental stages. The present study is the first comprehensive study of the stability of miRNA reference genes in Yellow River carp, providing valuable as well as basic data for investigating more accurate miRNA expression during gonadal differentiation and development of carp.