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. 2017 Aug 15;18(1):617.
doi: 10.1186/s12864-017-3974-1.

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress

Arabinda Mahanty  1   2 , Gopal Krishna Purohit  2 , Sasmita Mohanty  3 , Nihar Ranjan Nayak  4 , Bimal Prasanna Mohanty  5
Affiliations

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress

Arabinda Mahanty et al. BMC Genomics. .

Abstract

Background: High ambient temperature is known to affect fish gonadal development and physiology in a variety of ways depending on the severity and duration of exposure; however, the underlying molecular mechanisms are poorly understood. Gonadal gene expression influence the gonadal development, physiology and the quality of egg/sperm produced in teleosts and the mechanistic understanding of spatio-temporal changes in the gonadal gene expression could be instrumental in controlling the fate of egg/sperm and the quality of seed produced. Real time-quantititative polymerase chain reaction (RT-qCR), is a high throughput, sensitive and reproducible methodology used for understanding gene expression patterns by measuring the relative abundance of mRNA transcripts. However, its accuracy relies upon a suitable reference gene whose expression levels remain stable across various experimental conditions. In the present study, we evaluated the suitability of ten potential reference genes to be used as internal controls in RT-qPCR analysis in gonadal tissues (ovary and testis) of minnow Puntius sophore exposed to high temperature stress for different time periods (7 days, 60 days). Expression analysis of ten different constitutively expressed genes viz. 18S ribosomal RNA (18S rRNA), beta actin (βactin), β-2 microglobulin (b2mg), eukaryotic elongation factor-1 (eef1), glyceraldehyde-3phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), ribosomal binding protein L13 (rpl13), tubulin (tub), tata box binding protein (tbp), ubiquitin (ubi) was carried out by using RT-qPCR and the stability in their expressions were evaluated by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder.

Results: In ovary, eef1 was found to be the most suitable reference gene in all the algorithms used. In testis, b2mg was found to be the most suitable reference gene in delta Ct, BestKeeper, NormFinder analysis while tbp and eef1 were found to be the most suitable reference genes in geNorm analysis.

Conclusions: In conclusion, eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively, of Puntius sophore exposed to high temperature stress, and could be used as internal controls for gene expression analysis in gonadal tissues of Puntius sophore.

Keywords: Puntius sophore; RT-qPCR; Reference genes; Reproductive physiology; Thermal stress.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Stability in expression of 10 reference genes in a ovary and b testis of Puntius sophore as determined by delta Ct method. eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively
Fig. 2
Fig. 2
geNorm stability values of the 10 reference genes in a ovary and b testis of Puntius sophore. In ovary, eef1 and tubulin were the most stable genes whereas in testis b2mg and tbp were the most suitable reference genes. geNorm stability values (M) for each genes was calculated as the average pairwise variation for that gene with all other tested reference genes using the genorm software and genes were ranked in the increasing order of stability values
Fig. 3
Fig. 3
Normfinder stability values of the 10 reference genes in a ovary and b testis of Puntius sophore. In ovary, eef1 was the most stable gene whereas in testis b2mg and tbp were the most suitable reference genes as determined by the Normfinder algorithm. Normfinder stability values were calculated by combining the inter and intra group variations determined using the excel-Normfinder add-in. The genes were then ranked in the increasing order of stability values
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References

    1. Fujimoto T, Nishimuraa T, Goto-Kazetob R, Kawakamib Y, Yamahab E, Arai K. Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish. Proc Natl Acad Sci U S A. 2010;107:17211–17216. doi: 10.1073/pnas.1007032107. - DOI - PMC - PubMed
    1. Migaud H, Bell G, Cabrita E, McAndrew B, Davie A, Bobe J, Herraez MP, Carrillo MM. Gamete quality and brood stock management in temperate fish. Rev Aquacult. 2013;5(Suppl. 1):S194–S223. doi: 10.1111/raq.12025. - DOI
    1. Budd AM, Banh QQ, Domingos JA, Jerry DR. Sex control in fish: approaches, challenges and opportunities for aquaculture. J Mar Sci Eng. 2015;3:329–355. doi: 10.3390/jmse3020329. - DOI
    1. Valdebenito II, Gallegos PC, Effer BR. Gamete quality in fish: evaluation parameters and determining factors. Zygote. 2015;23:177–197. doi: 10.1017/S0967199413000506. - DOI - PubMed
    1. Sullivan CV, Chapman RW, Reading BJ, Anderson PE. Transcriptomics of mRNA and egg quality in farmed fish: some recent developments and future directions. Gen Comp Endocrinol. 2015;221:23–30. doi: 10.1016/j.ygcen.2015.02.012. - DOI - PubMed