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.
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.
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. welsicau@126.com.
Erlong
E
Wang
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. kywangsicau@126.com.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. kywangsicau@126.com.
Kaiyu
K
Wang
Department of Aquaculture, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. chendf_sicau@126.com.
Defang
D
Chen
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. wangjun1986616@gmail.com.
Jun
J
Wang
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. heyang@sicau.edu.cn.
Yang
Y
He
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. lbsicau@126.com.
Bo
B
Long
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. yangleigy@163.com.
Lei
L
Yang
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. yangqiansicau@126.com.
Qian
Q
Yang
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. gengyisicau@126.com.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. gengyisicau@126.com.
Yi
Y
Geng
Department of Aquaculture, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. hxldyq@126.com.
Xiaoli
X
Huang
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. ouyang.ping@live.cn.
Ping
P
Ouyang
Department of Basic Veterinary, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. nwm_mm2004@163.com.
Weimin
W
Lai
D000818:Animals; D005393:Fish Diseases / Q000378:metabolism / Q000517:prevention & control; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000592:standards*; D007075:Immunoglobulin M / Q000235:genetics / Q000378:metabolism; D017053:Infection Control; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012016:Reference Values; D017210:Tilapia / Q000235:genetics* / Q000276:immunology; D014611:Vaccination
D029941:Fish Proteins; D007075:Immunoglobulin M