Sample collection and genotyping
For each subject, a 5 ml peripheral blood sample was collected in EDTA
anticoagulant tubes. The sample was centrifuged, and then the plasma was
replaced with an equal amount of physiological solution (0.9% sodium
chloride) and stored at -80°C. Genomic DNA was extracted from whole
blood using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA), in
accordance with the manufacturer’s instructions. For the evaluation of
selected SNPs in angiogenesis-related genes, DNA samples of 34 HCC
patients were genotyped using the TaqMan allelic discrimination method
(StepOne Plus™ Real-Time PCR System, Applied Biosystems, Monza, Italy)
using commercial (VEGF-A rs2010963, VEGF-C rs4604006, HIF-1Α rs12434438,
ANGP2 rs55633437, NOS3 rs2070744) genotyping assays (ThermoFisher
Scientific, Waltham, MA, USA). DMET Plus assay (ThermoFisher Scientific,
Waltham, MA, USA.) was performed as previously
described.19-21