Validation of Quantitative Real Time PCR for JAK2 Mutation Detection by Using 2- ΔΔCt Method.
Rashmi Batra, Chih-Kang Huang, Qiulu Pan. St Luke's Roosevelt Hospital Center, University Hospital of Columbia University College of Physicians and Surgeons, New York, NY; Montefiore Medical Center, Albert Einstein College of Medicine, New York, NY
Background: The V617F mutation of the Janus kinase2 (JAK2) gene is a useful biomarker for the diagnosis of myeloproliferative disorders (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET) and myeloid metaplasia with myelofibrosis (MF). JAK2 V617F is a gain of function mutation that leads to clonal proliferation; it is present in about 95% of PV cases and 50% of ET and MF cases. The aim of this study was to compare 2 methods for JAK2 mutation detection such as restriction fragment length polymorphism (RFLP) and real time PCR with 2- ΔΔCt calculation.
Design: In a retrospective cohort study, 254 samples were analyzed for genomic DNA from blood or bone marrow with the RFLP assay and real time PCR. In RFLP, PCR is used to amplify the portion of JAK2 mutation bearing region. Amplicon products are then digested with the Bsa XI restriction enzyme which generates products of 30bp, 97bp and 140bp from a 267 bp amplicon. Samples harboring a V617F JAK2 mutation don't have the Bsa XI restriction enzyme leaving the 267 bp amplicon intact. Capillary electrophoresis is used to resolve the different sized amplicon products. Real time PCR using a mutation specific primer pair detects the absolute quantity by determining the copy number of the genomic DNA by using the 2- ΔΔCt calculation, in this study. The PCR cycle in which a threshold signal is generated above the background noise is called the threshold cycle (Ct). Positive result means that the Ct value of the tested sample is ≤40.
Results: Sensitivity of real time PCR is as low as 0.01% (1 positive cell per 10,000 cells) whereas that of RFLP is 5.0% (5 clonal cells in 100 normal cells). Out of 254 samples, 31 samples were positive with real time-PCR of which only 24 were positive with RFLP; that is 77.4% concordance between 2 methods. In the remaining 7 cases, the results were either normal or indeterminate on RFLP whereas the real time-PCT 2- ΔΔCt calculation in these subjects ranged from 0.02-0.4%. 4 cases with indeterminate results on RFLP were negative on real time-PCR.
Conclusions: This study suggests real time PCR as a more sensitive method for JAK2 mutation detection, which can also quantify the genomic DNA. The 2- ΔΔCt method reduces the time needed for preparation of standards and making a standard curve. In addition it reduces the contamination risk, reagent usage and the overall cost by reducing the number of reactions run each time.
Monday, February 28, 2011 9:15 AM
Platform Session: Section G 1, Monday Morning