Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass (Lateolabrax maculatus) under normal and salinity stress conditions

Haolong Wang; Haishen Wen; Yun Li; Kaiqiang Zhang; Yang Liu

doi:10.7717/peerj.5631

Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass (Lateolabrax maculatus) under normal and salinity stress conditions

PeerJ. 2018 Sep 19:6:e5631. doi: 10.7717/peerj.5631. eCollection 2018.

Authors

Haolong Wang^{1

2}, Haishen Wen^{1

2}, Yun Li^{1

2}, Kaiqiang Zhang^{1

2}, Yang Liu^{1

2}

Affiliations

¹ College of Fisheries, Ocean University of China, Qingdao, China.
² The Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Ocean University of China, Qingdao, China.

Abstract

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 (Lateolabrax maculatus), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes (HPRT, GAPDH, EF1A, TUBA, RPL7, RNAPol II, B2M, ACTB and 18S rRNA) were analyzed by qRT-PCR in 10 tissues (intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen) of L. maculatus. 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 18S rRNA was most stable in different tissues under normal conditions. During salinity stress, RPL7 was the most stable gene according to overall ranking and the best combination of reference genes was RPL7 and RNAPol II. In contrast, GAPDH 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 L. maculatus by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in L. maculatus. The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.

Keywords: Expression stability; Lateolabrax maculatus; Reference genes; qRT-PCR.

Grants and funding

The present study was financially supported by the National Natural Science Foundation of China (NSFC, 31602147), Shandong Provincial Natural Science Foundation, China (ZR2016CQ21), the key laboratory of Mariculture (KLM), Ministry of Education, OUC (NO:2018008) and China Agriculture Research System (CARS-47). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.