Fig. 5

Stability of selected reference transcripts according to the algorithms of geNormPlus and NormFinder. Stability analysis was performed in cohort II (n = 15). The x-axis presents the ranking of reference transcripts in order of increasing stability from left to right. Transcripts with the highest stability value exhibit the least stable expression level, while those with the lowest stability values are the most stable, respectively. NormFinder stability values are listed in ascending order (a). The most stable (top four) transcripts are highlighted by black squares: HPRT1, PPIA, PGK1 and GUSB. geNormPlus analysis shows the calculation of the average expression stability M-value of selected reference transcripts determined by RT-qPCR. High stability is defined by an M-value of < 0.5 as indicated by the dotted line (b). PGK1, PPIA, GUSB and HPRT1 displayed a M-value < 0.5 (black squares). In addition, geNormPlus calculates the optimal number of reference genes, taking into account the variable V as the average pairwise variation between two sequential candidate reference transcripts (c). The ratio V of the best two (V2) and best three (V3) was below 0.15, indicating that the best two reference genes [PGK1 and PPIA; enlarged black squares (b)] are sufficient