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

To meet these challenges of accurate interpretation of realtime qRT-PCR data, the authors suggested that an index of the most stable housekeeping genes should be used for normalization, and developed the geNorm VBA applet for Microsoft Excel in this regard [5] . A similar software tool, the BestKeeper, has been developed by Pfaffl et al. [6] . These tools can be used to find the most stable reference genes under different experimental conditions. We used the geNorm software which determines the individual stability of a gene within a pool of genes [5] . The stability is calculated according to the similarity of their expression profile by a pair-wise comparison, using their geometric mean as a normalizing factor. The gene with the highest M, i.e. the least stable gene, is then suggested excluded in a stepwise fashion until the most stable genes are determined, and an index suggested, based on the best genes. geNorm has been used to select the most stable reference genes in several recent studies (e.g. [4, 17, 18] ).