Expression levels of JAK2-V617F in granulocytes from human patients with MPD. (A) Expression of total JAK2 mRNA was determined by Taqman real-time PCR in purified granulocytes from healthy controls (n = 11) and patients with essential thrombocythemia (ET; n = 25), polycythemia vera (PV; n = 49), and primary myelofibrosis (PMF; n = 8). Values were normalized to the expression levels of β-glucuronidase (GUSB) mRNA. One control was chosen as the calibrator, and expression of JAK2 set to the value of 1 to calculate the fold expression in all other samples (ΔΔCT method). Boxes represent the interquartile range that contains 50% of the values, the horizontal line in the box marks the median and bars indicate the range of values. All significant differences with P < .05 (pairwise Mann-Whitney tests) are marked with asterisks (control vs PV: P = .0084, control vs PMF: P = .0003, ET vs PV: P = .026, ET vs PMF: P = .0197). Note that the expression values are shown on a logarithmic scale. (B) JAK2-V617F mRNA (left) and wild-type JAK2 mRNA (right) were quantified with allele-specific Taqman real-time assays, and the fold expression was calculated based on the values shown in panel A. JAK2-V617F expression was significantly higher in PV and PMF than in ET (ET vs PV: P < .0001, ET vs PMF: P = .0002, all other pairs: P > .05); n.d., not detectable. Wild-type JAK2 mRNA was significantly lower in PV than in ET or controls (control vs PV: P = .0026, ET vs PV: P = .0029). (C) Correlation between JAK2-V617F mRNA and JAK2-V617F DNA in patients with ET (■), PV (▴), and PMF (▾). The percentages of JAK2-V617F (%T = mutant JAK2 divided by total JAK2) in granulocyte RNA were plotted against the percentages of JAK2-V617F alleles in granulocyte genomic DNA for all patients studied. A strong linear correlation was noted (linear regression, r² = 0.97). (D) Ratios of mutant to wild-type JAK2 expression in granulocyte RNA calculated from the values shown in B (ET vs PV: P < .0001, ET vs PMF: P = .001).