Ex vivo expression analysis of Bsal reference gene candidates. Shown is the variation in the mRNA expression (Cq) of GAPDH, TUB, R6046 and α-centractin in (a–c) 5 × 106 Bsal spores and (d–f) 1 × 107 Bsal spores that were incubated for 2 hours at 15 °C (c,f) with or (b,e) without skin tissue of fire salamanders, or (a,d) a combination of both. Every condition was tested in fivefold. The whiskers represent the median, the minimum and maximum values, and the first and third quartiles. A plus (+) indicates the mean Cq value. (G) GeNorm stability analysis providing a GeNorm M and coefficient of variation (CV) value, combined with NormFinder intergroup variation analysis.