Wang Haolong, Wen Haishen, Li Yun, Zhang Kaiqiang, Liu Yang
College of Fisheries, Ocean University of China, Qingdao, China.
The Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Ocean University of China, Qingdao, China.
PeerJ. 2018 Sep 19;6:e5631. doi: 10.7717/peerj.5631. eCollection 2018.
The aim of this study was to select the most suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) of spotted sea bass (), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes (, , , , , , , and ) were analyzed by qRT-PCR in 10 tissues (intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen) of . Four algorithms, geNorm, NormFinder, BestKeeper, and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. The results showed the was most stable in different tissues under normal conditions. During salinity stress, was the most stable gene according to overall ranking and the best combination of reference genes was and . In contrast, was the least stable gene which was not suitable as reference genes. The study showed that different algorithms might generate inconsistent results. Therefore, the combination of several reference genes should be selected to accurately calibrate system errors. The present study was the first to select reference genes of by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in . The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.
本研究的目的是在正常生理条件和盐度胁迫条件下，为亚洲太平洋地区一种重要的商业海水鱼类——花鲈的定量实时聚合酶链反应（qRT-PCR）选择最合适的内参基因。通过qRT-PCR对花鲈10个组织（肠、肌肉、胃、脑、心脏、肝脏、鳃、肾脏、胸鳍和脾脏）中的9个候选内参基因（、、、、、、、和）进行了分析。采用geNorm、NormFinder、BestKeeper和比较ΔCt法四种算法评估候选内参基因的表达稳定性。结果表明，在正常条件下，在不同组织中最稳定。在盐度胁迫期间，根据综合排名，是最稳定的基因，最佳内参基因组合是和。相比之下，是最不稳定的基因，不适合作为内参基因。研究表明，不同算法可能产生不一致的结果。因此，应选择多个内参基因的组合来准确校准系统误差。本研究首次通过qRT-PCR筛选花鲈的内参基因，为花鲈选择合适的内参基因提供了有用的依据。本研究对该物种或其他硬骨鱼类的基因表达和功能基因组学研究也具有重要意义。
