Selection of normalization factors for quantitative real time RT-PCR studies in Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) under conditions of viral infection

Disease outbreaks caused by iridoviruses are known to affect farmed flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). To facilitate quantitative real time RT-PCR (qRT-PCR) analysis of gene expression in flounder and turbot during viral infection, we in this study examined the potentials of 9 housekeeping genes of flounder and turbot as internal references for qRT-PCR under conditions of experimental infection with megalocytivirus, a member of the Iridoviridae family. The mRNA levels of the 9 housekeeping genes in the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of flounder and turbot were determined by qRT-PCR at 24 h and 72 h post-viral infection, and the expression stabilities of the genes were determined with geNorm and NormFinder algorithms. The results showed that (i) viral infection induced significant changes in the mRNA levels of the all the examined genes in a manner that was dependent on both tissue type and infection stage; (ii) for a given time point of infection, stability predictions made by the two algorisms were highly consistent for most tissues; (iii) the optimum reference genes differed at different infection time points at least in some tissues; (iv) at both examined time points, no common reference genes were identified across all tissue types. These results indicate that when studying gene expression in flounder and turbot in relation to viral infection, different internal references may have to be used not only for different tissues but also for different infection stages.