Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder (Paralichthys olivaceus) is an important economic fish species cultured worldwide. In this report, we compared the potentials of ten housekeeping genes as quantitative real time RT-PCR (qRT-PCR) references for the study of gene expression in Japanese flounder under normal physiological conditions and during bacterial infection. For this purpose, the expression of the ten genes in eight flounder tissues (liver, spleen, kidney, heart, muscle, brain, gill, and intestine) was determined by qRT-PCR before and after bacterial infection. The expression levels of the housekeeping genes were then compared and evaluated with geNorm and NormFinder algorithms. The results showed that before bacterial infection, the tested genes exhibited tissue-specific expressions to various degrees, with β-actin and ubiquitin-conjugating enzyme being ranked as the most stable genes across tissue types. Following bacterial challenge, all the tested genes varied in expression levels in tissue-dependent manners and no cross-all-tissue type reference gene was identified among the examined panel of housekeeping genes; however, α-tubulin was recognized as the most stable gene in four (spleen, heart, muscle, and gill) of the eight examined tissues. These results indicate that for qRT-PCR analysis of gene expression in Japanese flounder as a function of bacterial infection, the choice of reference genes should be made according to tissue type.