定量实时逆转录酶聚合酶链反应(RT-qPCR)已被广泛用于研究与鱼类免疫学相关的基因表达。在这样的研究中,应选择稳定的参考基因来纠正目标基因的表达。在这项研究中,七个候选参考基因(甘油醛-3-磷酸脱氢酶(GADPH),泛素结合酶(UBCE),18S核糖体RNA(18SrRNA),β-2-微球蛋白(B2M),伸长因子1α(EF1A),微管蛋白α链样(TUBA)和β肌动蛋白(ACTB),选择分析它们在七个组织中的稳定性和正常化(肝脏,脾,脾肾,大脑,心,尼罗罗非鱼(Oreochromisniloticus)的肌肉和肠)受到无乳链球菌或链球菌的攻击,分别。结果表明,所有候选参考基因均表现出组织依赖性转录变异。用PBS注射作为对照,UBCE是肠道内最稳定、最适宜的单一内参基因,肝脏,大脑,肾,S.iniae感染后的脾脏,在肝脏中,肾,无乳链球菌感染后的脾脏。EF1A在S.iniae或无乳链球菌感染后的心脏和肌肉中最合适。GADPH是无乳链球菌感染后肠和脑中最合适的基因。在正常情况下,UBCE和18SrRNA是在各种组织中表达最稳定的基因。这些结果表明,对于罗非鱼的RT-qPCR分析,选择两个或多个参考基因可能更适合于基因表达的跨组织分析。
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.