Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

Olsvik, Pål A; Lie, Kai K; Jordal, Ann-Elise O; Nilsen, Tom O; Hordvik, Ivar

The ideal endogenous control should be expressed at a constant level among different tissues of an organism, at all stages of development and should be unaffected by the experimental treatment. It should also be expressed at roughly the same level as the RNA under study [1] . However, data normalization in real-time RT-PCR remains a real problem, especially for absolute quantification [1] . Numerous studies have revealed that no single universal gene has a constant expression level under all developmental or experimental situations. The best choice of reference gene to use as an endogenous control varies, depending on the tissues of interest in the experiment. A large number of genes have for this reason been selected for normalization of mRNA expression data [2, 3] . If the selected reference gene fluctuates randomly between samples, small differences in expression between the genes of interest will be missed. Gene expression coefficient of variation (CV) between different groups of individuals should ideally be as low as possible [4] . In general, the stability of several potential reference genes should be tested in every examined tissue or cell, and under different experimental design [5, 6] . An increasing number of papers are discussing the selection of reference genes in real-time RT-PCR analyses [3, 7] .