Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus)

• Published in: Gene Vol. 527; no. 1; pp. 183 - 192
• Main Authors: Yang, Chang Geng, Wang, Xian Li, Tian, Juan, Liu, Wei, Wu, Fan, Jiang, Ming, Wen, Hua
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.09.2013
• Subjects: actin; Animals; brain; Cichlids - genetics; Cichlids - immunology; Cichlids - metabolism; Cichlids - microbiology; Fish Diseases - immunology; Fish Diseases - metabolism; Fish Diseases - microbiology; Fish Proteins - genetics; Fish Proteins - metabolism; Gene Expression; Gene Expression Profiling - standards; genes; Genes, Essential; glyceraldehyde-3-phosphate dehydrogenase; heart; immunology; kidneys; liver; muscles; Oreochromis niloticus; Organ Specificity; Real-Time Polymerase Chain Reaction - standards; Reference genes; Reference Standards; reverse transcriptase polymerase chain reaction; ribosomal RNA; RT-qPCR; spleen; Streptococcal Infections - immunology; Streptococcal Infections - metabolism; Streptococcal Infections - veterinary; Streptococcus agalactiae; Streptococcus iniae; Tilapia; transcription (genetics); Transcriptome; tubulin; ubiquitin-protein ligase; PBS; R2; GADPH; E; Reference genes; TUBA; CTSZ; UBCE; Tilapia; ACTB; LPS; EF1A; Ct; V; 18S rRNA; LTA; NF; B2M; CTSD; RT-qPCR; beta actin; PCR efficiency; tubulin alpha chain-like; phosphate-buffered saline; cathepsin Z; R; pairwise variation; elongation factor 1 alpha; beta-2-microglobulin; Lipopolysaccharide; 18S ribosomal RNA; glyceraldehyde-3-phosphate dehydrogenase; cathepsin D; ubiquitin-conjugating enzyme; cycle threshold; correlation coefficients; sequential normalization factor; lipoteichoic acid
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2013.06.013

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. •The seven genes were examined to serve as qRT-PCR reference genes in tilapia.•Candidate reference genes exhibited tissue-dependent transcriptional variations.•Selecting two or more reference genes may be a more feasible way.