Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii
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.
KeywordTrachinotus blochii housekeeping gene expression stability reference gene
Unable to display preview. Download preview PDF.
- Amal M N A, Zamri-Saad M, Iftikhar A R, Siti-Zahrah A, Aziel S, Fahmi S. 2012. An Outbreak of Streptococcus agalactiae Infection in Cage-Cultured Golden Pompano, Trachinotus blochii (Lacépède), in Malaysia. Journal of Fish Diseases, 35 (11): 849–852, https://doi.org/10.1111/j.1365-2761.2012.01443.x.Google Scholar
- Andersen C L, Jensen J L, Ørntof T F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 64 (15): 5 245-5 250, https://doi.org/10.1158/0008-5472.CAN-04-0496.Google Scholar
- Dang W, Sun L. 2011. Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus). Fish & Shellfish Immunology, 30 (2): 720–728, https://doi.org/10.1016/j.fsi.2010.12.028.CrossRefGoogle Scholar
- Fernandes J M O, Mommens M, Hagen Ø, Babiak I, Solberg C. 2008. Selection of suitable reference genes for realtime PCR studies of Atlantic halibut development. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 150 (1): 23–32, https://doi.org/10.1016/j.cbpb.2008.01.003.CrossRefGoogle Scholar
- Haller F, Kulle B, Schwager S, Gunawan B, Von Heydebreck A, Sültmann H, Füzesi L. 2004. Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization. Analytical Biochemistry, 335 (1): 1–9, https://doi.org/10.1016/j.ab.2004.08.024.CrossRefGoogle Scholar
- Hattingh J. 1977. The effect of tricaine methanesulphonate (MS-222) on the microhaematocrit of fish blood. Journal of Fish Biology, 10 (5): 453–455, https://doi.org/10.1111/j.1095-8649.1977.tb04077.x.CrossRefGoogle Scholar
- Li D, Wu P, He M F, Li W, Xiao T Y, Chu W Y. 2016. Screening of reference genes in Siniperca chuatsi for qRT-PCR analysis. Life Science Research, 20 (3): 213–217, https://doi.org/10.16605/j.cnki.1007-7847.2016.03.005. (in Chinese with English abstract)Google Scholar
- Li Z J, Yang L J, Wang J, Shi W C, Pawar R A, Liu Y M, Xu C G, Cong W H, Hu Q R, Lu T Y, Xia F, Guo W, Zhao M, Zhang Y Y. 2010. β - Actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum. Fish & Shellfish Immunology, 29 (1): 89–93, https://doi.org/10.1016/j.fsi.2010.02.021.CrossRefGoogle Scholar
- Liu M, Chen X, Yang S Y. 2014. Marine Fishes of Southern Fujian, China, Volume 2. Ocean Press, Beijing, China. p.165–167. (in Chinese)Google Scholar
- Øvergård A C, Nerland A H, Patel S. 2010. Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes. BMC Molecular Biology, 11: 36, https://doi.org/10.1186/1471-2199-11-36.CrossRefGoogle Scholar
- Pfaffl M W, Tichopad A, Prgomet C, Neuvians T P. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeperexcel- based tool using pair-wise correlations. Biotechnology Letters, 26 (6): 509–515, https://doi.org/10.1023/B:BILE.0000019559.84305.47.CrossRefGoogle Scholar
- Qiu R, Sun B G, Fang S S, Sun L, Liu X. 2013. Identification of normalization factors for quantitative real-time RTPCR analysis of gene expression in Pacific abalone Haliotis discus hannai. Chinese Journal of Oceanology and Limnology, 31 (2): 421–430, https://doi.org/10.1007/s00343-013-2221-0.CrossRefGoogle Scholar
- Schoettger R A, Julin A M. 1967. Efficacy of MS-222 as an anaesthetic on four salmonids. In: Investigations in Fish Control, Resource Publication 19. U.S. Department of the Interior, Bureau of Sport Fisheries and Wildlife, Washington, DC, p.3–15.Google Scholar
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Roy N V, De Paepe A, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3 (7): RESEARCH0034, https://doi.org/10.1186/gb-2002-3-7-research0034.