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

BMC Genomics

Research Article

Bimal Prasanna Mohanty¹  Arabinda Mahanty²  Gopal Krishna Purohit³  Sasmita Mohanty³  Nihar Ranjan Nayak⁴ 

[1] Fishery Resource and Environmental Management Division, ICAR- Central Inland Fisheries Research Institute, Barrackpore, 700120, Kolkata, India;Fishery Resource and Environmental Management Division, ICAR- Central Inland Fisheries Research Institute, Barrackpore, 700120, Kolkata, India;School of Biotechnology, KIIT University, 751024, Bhubaneswar, Odisha, India;School of Biotechnology, KIIT University, 751024, Bhubaneswar, Odisha, India;Wayne State University Perinatal Initiative, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 48201, Detroit, MI, USA;

关键词: Reference genes;    RT-qPCR;    Reproductive physiology;    Thermal stress;    Puntius sophore;   

DOI  :  10.1186/s12864-017-3974-1

 received in 2016-10-31, accepted in 2017-07-31,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundHigh 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.ResultsIn 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.ConclusionsIn 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.

【 授权许可】

CC BY   
© The Author(s). 2017