Real-Time Quantitative PCR

Quantitative real-time PCR analyses of human VEGF gene was performed using a LightCycler 480 (Roche Diagnostics, Roche Applied Science, Mannheim, Germany) and Taqman oligonucleotide probe 5^’ CCA AGT GGT CCC AGG CTG CAC C 3^’ and forward 5^’ TTG CTG CTC TAC CTC CAC CAT 3^’ and reverse 5^’ CAC TTC GTG ATG ATT CTG CCC 3^’ primers. The oligonucleotide probes were fluorescently labeled on the 5^’ end with FAM (6-carboxy fluorescein) and on the 3^’ end with BlackBerry Quencher (BBQ). Quantitative real-time PCR analyses of human HIF-1α gene was performed using oligonucleotide probe 5^’ AGC AAC AGG GAA AGC GTG GCT 3^’ and forward 5^’ GGC AGG AAG ATT GTC ATG GAC 3^’ and reverse 5^’ TCT GTC TGT CAC ATG GGT GAT GAA 3^’ primers (TIB MOLBIOL GmbH, Berlin, Germany). Real-time quantitative PCR of eNOS gene was performed on a Mastercycler EP RealPlex (Eppendorf AG, Hamburg, Germany) using the Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, Cambridge, UK) and forward: 5^’ CGG CAT CAC CAG GAA GAA GA 3^’ and reverse 5^’ GCC ATC ACC GTG CCC AT 3^’ primers. β-actin was used as an internal control for normalization of the examined angiogenic factors. Real-time PCR was performed in granulocytes of total 83 MPN patients, but because of overlapping (samples from the same patient): HIF-1α was evaluated in 50 samples (20 PV, 15 ET, 15 PMF, plus 6 controls), VEGF in 52 samples (20 PV, 16 ET, 16 PMF, plus 6 controls) and eNOS in 58 samples (20 PV, 23 ET, 15 PMF, plus 6 controls). Real-time PCR was performed in CD34⁺ cells of total 57 MPN patients, but because of overlapping: HIF-1α was evaluated in 36 samples (16 PV, 11 ET, 9 PMF, plus 5 controls), VEGF in 44 samples (22 PV, 11 ET, 11 PMF, plus 5 controls), and eNOS in 39 samples (15 PV, 15 ET, 9 PMF, plus 5 controls).