Abstract
Trachinotus blochii is one of the important commercial fish species. In this study, we aim to confirm the reliability reference genes in T. blochii during different bacterial challenge through quantitative real-time PCR (qRT-PCR). The expression of the seven selected genes in four immune organs (i.e., spleen, kidney, intestine, and gill) stimulated with Vibrio harveyi, Edwardsiella tarda, and Streptococcus agalactiae were determined by qRT-PCR. The PCR data was analyzed using the geNorm and NormFinder algorithms. The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation. After 48 h of stimulation with V. harveyi, geNorm ranked EF1A/Actin, 18S rRNA/B2M, UBCE/B2M, and 18S rRNA/B2M, as the most stably expressed genes in spleen, kidney, intestine, and gill, respectively. After 48 h of stimulation with E. tarda, geNorm ranked 18S rRNA/EF1A, 18S rRNA/B2M, B2M/RPL13, and 18S rRNA/EF1A, as the most stably expressed genes in spleen, kidney, intestine, and gill, respectively. After 48 h of stimulation with S. agalactiae, 18S rRNA/ EF1A, 18S rRNA/B2M, B2M/Actin, and 18S rRNA/B2M were ranked as the most stably expressed genes in spleen, kidney, intestine, and gill, respectively. Compared to the results analyzed by geNorm, reference genes received similar rankings when using NormFinder software. The results showed that the reference genes appeared to be not only tissue specific, but also specific to the infecting species of bacteria. If one gene is preferred when T. blochii were infected by bacteria, 18S rRNA, B2M, B2M, 18S rRNA may be used in spleen, kidney, intestine, and gill, respectively.
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Data Availability Statement
The authors declare that all data supporting the findings of this study are available within the article.
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Supported by the National Natural Science Foundation of China (No. 41666006, 31702379) and the Nanhai Famous Youth Project
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Chen, X., Zhang, X., Sun, Y. et al. Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii. J. Ocean. Limnol. 38, 480–489 (2020). https://doi.org/10.1007/s00343-019-9053-5
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DOI: https://doi.org/10.1007/s00343-019-9053-5