AID mRNA expression levels in patients with mutated and unmutated CLL. Quantitative PCR for AID and GAPDH as the endogenous control was performed on PBMCs from patients with MUT and UM CLL. CLL B cells stimulated with CD40L/IL-4, tonsil samples, and PBMCs from 8 healthy donors were used as positive and negative calibrators, respectively. By subtraction of the mean threshold cycle (Ct) triplicate AID measurements with the mean Ct from triplicate GAPDH measurements, the mean ΔCt was calculated. The ΔΔCt values were calculated with the mean ΔCt of the 8 healthy donors and the 3 independently experiments of CLL B cells activated with CD40L/IL-4 as calibrators. AID negativity was defined by the absence of AID expression in duplicate analysis. The expression factor difference and range were calculated by the following formulas: 2^−ΔΔCt (mean factor difference); 2^{−(ΔΔCt − ΔCt SD)} and 2^{−(ΔΔCt + ΔCt SD)} (error bars indicate range factor difference). The factor difference conversion of the ΔΔCt is depicted in the graph in relative percentages of AID expression.