Figure 4

Normalization bias analysis according to reference genes selection. (A) Example of normalized transcript levels of abf-B2 and xynC under different culture conditions (Thio-gentiobiose, Arbocel and C starvation samples) using 4 different Normalization Factors (NFs): NF_{(R2, R10, R6)}, NF_(R5,R11,R3), NF_(R3) and NF_(β-tub). Log _{(base 2)} of FC values on the Y axis (mean ± SD, n = 2 in this experiment) using glucose as the calibrator sample. (B) Comparison of NF_{(R2, R10, R6)} to NF calculated from less stable genes, as well as from single genes such as R3 and β-tub. For each condition of interest (X axis, see Additional file 1), we calculated a normalization bias (i.e. under- or over-estimation of the normalised expression value of GOIs) as the ratio between the theoretically best NF (NF_{(R2, R10, R6)} as determined from geNorm classification by using the entire set of conditions) and NF calculated from other combinations of reference genes. Log _{(base 2)} of the normalization bias is represented on the Y axis. Yellow zone: less than 1.5 fold bias; Green zone: 1.5─2 fold bias; Blue zone: 2─3 fold bias; Red zone: 3─8 fold bias. (C) Quantile plot of the normalization bias values for each NF. The normalization bias (Log _{(base 2)}) is represented on the X axis and the same colour code used in (B) was applied. The quantile fractions are represented on the Y axis. (♦) NF_(R12,R7,R8), (☐) NF_(R4,R9,R1), (◯) NF_(R5,R11,R3), (+) NF_(R3) and (×) NF_(β-tub).