Screening of stable internal reference genes by quantitative real-time PCR in humpback grouper Cromileptes altivelis

Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value. To determine the expression stabilities of six commonly used internal reference genes in C. altivelis challenged by Vibrio harveyi and viral nervous necrosis virus (VNNV) through quantitative real-time PCR (qRT-PCR), the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm, NormFinder, and BestKeeper. The results show that the expression stabilities of the six candidate internal reference genes were different. Under normal physiological conditions, RPL13 were identified as the most stably expressed genes among five different immune organs (liver, spleen, kidney, intestine, and gill). After V. harveyi stimulation, RPL13, RPL13, EF1A, RPL13, and EF1A were identified by geNorm, NormFinder, and BestKeeper as the most stable genes in liver, spleen, kidney, intestine, and gill, respectively. Combining these three algorithms suggested that under stimulation of VNNV, RPL13, EF1A, Actin, RPL13, and Actin were as the most stable genes in liver, spleen, kidney, intestine, and gill, respectively. These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis. This study provided a solid foundation for future studies on gene expression of C. altivelis under different conditions.