Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, 150070, China.
Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture and Rural Affairs, Harbin, 150070, China.
Sci Rep. 2022 Jan 10;12(1):313. doi: 10.1038/s41598-021-03872-x.
As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.
作为一种强大而有吸引力的基因表达检测方法，qRT-PCR 已广泛应用于水产养殖研究。由于其生态和经济价值，人们对哲罗鱼（Hucho taimen）的生物学越来越感兴趣。为了可靠地定量基因表达，需要稳定的参考基因，但哲罗鱼的这种基因尚未得到优化。在这项研究中，使用 geNorm、NormFinder、BestKeeper 和 RefFinder 评估了 10 种常用候选参考基因的稳定性水平。使用 240 个样本检测了 10 个基因的表达水平，这些样本来自于 48 个实验组，每个实验组由 40 个个体组成，这些个体分别在 4 种热应激条件（18、20、22 和 24°C）下处理 24 小时和 26°C 下处理 4、24、48 和 72 小时。从每个个体中采集 6 种组织（血液、心脏、大脑、鳃、皮肤和肝脏）。核糖体蛋白 S29（RPS29）和核糖体蛋白 L19（RPL19）是所有样本中最稳定的基因，而 28S 核糖体 RNA（28S rRNA）、附着区结合蛋白（ARBP）和 18S 核糖体 RNA（18S rRNA）是最不稳定的基因。这些结果通过对哲罗鱼热应激基因（热休克蛋白 60、hsp60 和热休克蛋白 70、hsp70）的表达分析得到了验证。总之，RPS29 和 RPL19 是哲罗鱼 qRT-PCR 分析的最佳参考基因，无论组织和实验条件如何。这些结果使得哲罗鱼基因表达的可靠研究成为可能。
