MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets.

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.