Ranking, stability and determination of optimal number of reference genes using geNorm. A, B and C represent ranking based on average expression stability value ( M) for developmental stages, tissues and treatments. In the plots, genes were ordered from least (left) to most (right) stable. D, E and F display optimal number of genes required for the accurate estimation of the target gene mRNA in developmental stages, tissues and treatments calculated by the pairwise variation (V) of the normalization factors (NFn and NFn + 1). A large pairwise variation indicates that the added gene has a significant effect and should preferably be included in the normalization. The cut-off value for such significance should be 0.15.