![pbp3 gene rt-qpcr pbp3 gene rt-qpcr](https://ars.els-cdn.com/content/image/1-s2.0-S0022283610011320-fx1.jpg)
GADPH was the most suitable gene in intestine and brain after S.
![pbp3 gene rt-qpcr pbp3 gene rt-qpcr](https://www.researchgate.net/publication/343894231/figure/fig4/AS:936836474097672@1600370629208/RT-qPCR-verification-of-the-up-regulated-genes-involved-in-the-innate-immunity-by-DTMUV.png)
EF1A was the most suitable in heart and muscle after S. iniae infection, and in the liver, kidney, and spleen after S. 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. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. 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. In such studies, a stable reference gene should be selected to correct the expression of the target gene. Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology.