Fig. 4. RT-PCR of Fas and FasL in 530 melanoma cells under hypoxia. 530 melanoma cells were either kept under normoxia or exposed to 24 h of severe hypoxia. Jurkat cells stimulated with PHA and kept under normal culture conditions served as a positive control for FasL expression. Total RNA was extracted using a commercially available RNA extraction kit. Total RNA (1 µg) was reverse transcribed into cDNA using an oligo(dT)16 primer. For PCR amplification, specific primers for Fas and FasL were used. PCR conditions were as follows: an initial 5-min denaturation step was followed by 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 58°C, 1 min of primer extension at 72°C, and a final primer extension step of 10 min at 72°C. PCR products were visualized after electrophoresis on a 1% agarose gel.