Quantitative PCR Analysis of EPCs

For these experiments, we used Real 7900 HT PCR (as above), TaqMan Universal PCR Master Mix-4304437, Human GAPDH-Hs03929097_g1, and a 384-well PCR plate (Thermo Fisher Scientific, Waltham, MA, USA). Human EPCs (4 × 10⁴), untreated or DHT-treated (30 nM), were incubated as described for immunocytochemistry experiments. After 48 h, DNA was isolated using the PureLink Genomic DNA Isolation Kit (Life Technologies, Waltham, MA, USA) and quantified by the NanoDrop ND-1000 UV-VIS spectrophotometer. To accurately determine the relative cellularity, a calibration curve was established by serially diluting standardized DNA extracted from a reference sample of 4 × 10⁴ EPCs to a final dilution of 2500 equivalent cells [56 (link)]. In addition, for the design of oligonucleotide sequences, we compared the human genome to the mouse genome using the Genome ARTIST V1 software [57 (link)]. Experimental conditions for PCR were chosen according to the manufacturer’s specifications, and all experiments were performed in triplicate.