INTRODUCTION: Currently, the JAK2V617F allele quantification in Ph- MPN is performed on genomic DNA. However, compared to DNA, mRNA analysis may offer some advantages such as: 1)higher sensitivity; 2) inclusion of platelet mRNA; 3) mRNA transcriptional functionality. Therefore, we developed an absolute allele-specific RQ-PCR method to quantify JAK2WT, V617F and ABL (as housekeeping gene) transcript levels aiming to: 1) verify specificity and sensitivity of this assay; 2)evaluate whether gene expression levels correlate with disease phenotype and may mark disease progression (i.e. ET toward PV). METHODS: To construct reference curves, including five 10-fold serial dilutions, two plasmid standards were manufactured to contain 150 bp of either JAK2WT and JAK2V617F cDNA sequence. To perform an allelic discrimination we used primers described by Merker et al (JMD, 2010): reverse primers are complementary to the WT or the mutant cDNA sequence, respectively. Assay sensitivity was determined by serial 10-fold dilutions of the K562 (JAK2WT) and HEL (V671F homo) cell lines which were included in each experiment as negative and positive controls. RESULTS: We tested cDNA synthesized from peripheral blood buffy coat specimens of 48 MPN patients (16 PV; 25 ET, 7 PMF), 13 patients with secondary polycythemia (SP) and 23 healthy donors (CTRL). Respect to the qualitative PCR method (Baxter el al, Lancet 2005) our RQ-PCR assay shows a 100% concordance rate in identifying the presence of the JAK2V617F. V617F mutation was detected in 16/16 PV (100%), 19/25 ET (76%) and 5/7 (71%) PMF samples. The mean expression of JAK2WT allele, as absolute averaged copies+SEM was: healthy donors 12127+2181; SP 12244+2489; PV 4955+1642, WT ET 10876+1781; V617F ET 5693+830 and V617F PMF 3004+1442. Statistically significant differences were detected between CTRL and PV (p