2.10. Assay for mRNA Expression

The mRNA expression for VEGF, bFGF, TNF, IL-6 and IL-8 was examined by real-time RT-PCR. Poly A⁺ mRNA was isolated from 12 h-cultured cells using oligo (dT)-coated magnetic micro beads (Milteny Biotec). First-strand cDNA was synthesized from 1.0 mg of Poly A⁺ mRNA using a Superscript cDNA synthesis kit (Invitrogen Corp., Carlsbad, CA, USA) following the manufacturer’s instructions. A polymerase chain reaction (PCR) was carried out using the GeneAmp 5700 Sequence Detection System (Applied Biosystems, Forster City, CA, USA). The PCR mixture included 2.0 µL of the sample cDNA solution (100.0 ng/µL), 25.0 µL of SYBR-Green Mastermix (Applied Biosystems), 0.3 µL of both sense and antisense primers and distilled water, with a total volume of 50.0 µL. The reaction was conducted as follows: 4 min at 94 °C, followed by 40 cycles for 4 min at 95 °C, 1 min at 60 °C and 1 min at 70 °C [29]. β-actin was amplified as an internal control. The angiogenic factor mRNA levels were calculated by using the comparative parameter threshold cycle and were normalized to β-actin levels. The nucleotide sequences of the primers are shown in Table 1 [29,30,31].

The nucleotide sequences of the primers.

VEGF: vascular endothelial growth factor; bFGF: basic fibroblast growth factor; TNF: tumor necrosis factor-α.