Optimal number of reference genes. Pair wise variation of normalization factors after successive inclusion of less stable genes determined the optimal number of reference genes. On the left-most side is the pair wise variation when the number of genes is enlarged from 2 to 3 (V2/3). Stepwise inclusion of less stable genes generates the next data points. Inclusion of a 4th gene has a significant effect on the normalization factors. Inclusion of a 5th, 6th, or 7th gene has a negative effect on the pair wise variation value and reflects the average expression stability M of these genes.