Figure 2

Determination of the optimal number of reference genes as internal references for normalization using geNorm analysis. The geNorm program calculates a normalization factor from at least two reference genes and the mean pairwise variation (V) between every combination of sequential normalization factors in order to determine the minimum number of reference genes required for accurate normalization in the samples. For example, V 5/6 represents the comparison of the normalization factors from five and six reference genes, respectively. On the left-most side is the pairwise variation when the number of reference genes is increased from two to three (V 2/3). Stepwise inclusion of less stable genes generates the subsequent data points. A decrease in the V value indicates a positive effect and means that the added gene should preferably be included for calculation of a reliable normalization factor. The cutoff value for V, below which the inclusion of an additional reference gene does not result in a significant improvement of normalization, was set at 0.15. It was apparent from the analysis of all studied samples that the combination of the two most stable reference genes is the best option and the combination of the five most stable reference genes is the second-best option for accurate normalization