Laboratório de Genômica Estrutural, Programa de Pós-Graduação em Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Rio Grande, RS, Brazil.
Fish Physiol Biochem. 2023 Jun;49(3):409-423. doi: 10.1007/s10695-023-01190-9. Epub 2023 Apr 19.
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.
尼罗罗非鱼（Oreochromis niloticus）是世界上最重要的养殖鱼类之一，但罗非鱼养殖受到低温的严重影响。最近的研究表明，microRNAs（miRNAs）调节鱼类的耐寒性特征。一般来说，基于 qPCR 的方法是 miRNA 定量最简便、最准确的形式。然而，qPCR 数据严重依赖于适当的归一化。因此，本研究的目的是确定先前测试的、稳定表达的 miRNAs 是否受到尼罗罗非鱼急性冷应激的影响。为此，在尼罗罗非鱼的四个组织（血液、大脑、肝脏和鳃）中，在两种实验条件（急性冷应激和对照）下，评估了一个小核 RNA（U6）和六个候选参考 miRNA（miR-23a、miR-25-3、Let-7a、miR-103、miR-99-5 和 miR-455）。通过四种独立的方法（delta Ct 法、geNorm、NormFinder 和 BestKeeper）分析每个候选参考 miRNA 表达的稳定性。此外，使用 RefFinder 构建了共识综合稳定性排名。总体而言，miR-103 是本研究中最稳定的参考 miRNA，miR-103 和 Let-7a 是最佳的参考靶标组合。同样重要的是，Let-7a、miR-23a 和 miR-25-3 在不同的组织和实验组中始终保持稳定。考虑到所有变量，U6、miR-99-5 和 miR-455 在急性冷应激下是最不稳定的候选者。最重要的是，尼罗罗非鱼中验证了合适的参考 miRNA，为该物种进一步准确的 miRNA 定量提供了便利。
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