Reverse transcription quantitative (RT-q)PCR

Total RNA of cells (FBS-supplemented non-induced cells, hPL-supplemented non-induced cells, FBS-supplemented induced cells and hPL-supplemented induced cells; n=5) was extracted using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare Life Sciences). First strand complementary DNA (cDNA) was then synthesized from total RNA using a Tetro cDNA synthesis kit (cat. no. BIO-65043; Bioline), according to the manufacturer's instructions. Briefly, the samples were incubated at 45°C for 30 min, followed by 85°C for 5 min to terminate the reaction. Gene transcripts were measured using a SensiFAST™ SYBR^® No-ROX kit (cat. no. BIO-98005; Bioline) with a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR primers targeting von Willebrand Factor (vWF), VEGF receptor 2 (VEGFR2), and endothelial nitric oxide synthase (eNOS) were used (Table I) (17). The protocol consisted of 36 cycles of 30 sec of denaturation at 95°C, 30 sec of annealing at 60°C and 60 sec of extension at 72°C. GAPDH was used as an internal control. The expression level of endothelial specific genes was plotted using the 2^−ΔΔCq method (18).

Primer sequences of reverse transcription-quantitative PCR.

vWF, von Willebrand factor; VEGFR2, vascular endothelial growth factor receptor 2; eNOS endothelial nitric oxide synthase.