Evaluation of reference genes for quantitative real-time PCR analysis of gene expression in Hainan medaka (Oryzias curvinotus)

Quantitative real-time PCR (qRT-PCR) is a powerful and sensitive methodology to analyze expression of target genes. qRT-PCRs rely on selecting suitable reference gene. In the present study, 17 candidate reference genes in Hainan medaka, Oryzias curvinotus, were selected, and candidate genes were studied for mRNA expression levels and stabilities in different tissues and at different stages of embryonic development. Expression patterns were analyzed using RefFinder and geNorm to test for suitability of the gene as a reference gene. RefFinder analysis ranked the stability of the reference genes in the following order (Top 5): rps4x > eef1b > 60srp > dcakd > eef1a in different tissues, and mrp13 > rps20 > actb > sbds > adp in embryonic development. Similarly, geNorm selected eef1b and rps4x as the two optimal reference genes for normalizing target genes expression in Hainan medaka. The results demonstrated that the expression of reference genes is variable under different experimental conditions, highlighting the importance of validating the stability and optimal number of reference genes before doing the qRT-PCR experiment.