title: Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).
abstract: Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression.
authors.affiliation: Key Laboratory of Freshwater Biodiversity Conservation and Utilization of Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.
authors.forename: Chang Geng
authors.initials: CG
authors.lastname: Yang
authors.forename: Xian Li
authors.initials: XL
authors.lastname: Wang
authors.forename: Juan
authors.initials: J
authors.lastname: Tian
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Fan
authors.initials: F
authors.lastname: Wu
authors.forename: Ming
authors.initials: M
authors.lastname: Jiang
authors.forename: Hua
authors.initials: H
authors.lastname: Wen
mesh_terms: D000818:Animals; D023681:Cichlids / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D005393:Fish Diseases / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000592:standards*; D020043:Genes, Essential; D009928:Organ Specificity; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D013290:Streptococcal Infections / Q000276:immunology / Q000378:metabolism / Q000662:veterinary*; D059467:Transcriptome
chemical_list: D029941:Fish Proteins
keywords: 18S rRNA; 18S ribosomal RNA; ACTB; B2M; CTSD; CTSZ; Ct; E; EF1A; GADPH; LPS; LTA; Lipopolysaccharide; NF; PBS; PCR efficiency; R(2); RT-qPCR; Reference genes; TUBA; Tilapia; UBCE; V; beta actin; beta-2-microglobulin; cathepsin D; cathepsin Z; correlation coefficients; cycle threshold; elongation factor 1 alpha; glyceraldehyde-3-phosphate dehydrogenase; lipoteichoic acid; pairwise variation; phosphate-buffered saline; sequential normalization factor; tubulin alpha chain-like; ubiquitin-conjugating enzyme