Next Article in Journal
Instrumentation on Multi-Scaled Scattering of Bio-Macromolecular Solutions
Next Article in Special Issue
Identification of MicroRNA for Intermuscular Bone Development in Blunt Snout Bream (Megalobrama amblycephala)
Previous Article in Journal
Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo
Previous Article in Special Issue
Identification and Characterization of a PRDM14 Homolog in Japanese Flounder (Paralichthys olivaceus)
Article Menu
Issue 5 (May) cover image

Export Article

Open AccessArticle
Int. J. Mol. Sci. 2015, 16(5), 9998-10015; doi:10.3390/ijms16059998

Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection

1
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China
2
Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China
3
Department of Aquaculture, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China
*
Author to whom correspondence should be addressed.
Academic Editor: Li Lin
Received: 10 March 2015 / Revised: 23 April 2015 / Accepted: 24 April 2015 / Published: 30 April 2015
(This article belongs to the Special Issue Fish Molecular Biology)
View Full-Text   |   Download PDF [1303 KB, uploaded 30 April 2015]   |  

Abstract

qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA. View Full-Text
Keywords: real time quantitative PCR; reference gene; stability; normalization; tilapia; rSip; Streptococcus agalactiae; IgM real time quantitative PCR; reference gene; stability; normalization; tilapia; rSip; Streptococcus agalactiae; IgM
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Wang, E.; Wang, K.; Chen, D.; Wang, J.; He, Y.; Long, B.; Yang, L.; Yang, Q.; Geng, Y.; Huang, X.; Ouyang, P.; Lai, W. Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection. Int. J. Mol. Sci. 2015, 16, 9998-10015.

Show more citation formats Show less citations formats

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top