Taqman universal pcr master mix 4304437

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.

RNA was extracted from cell pellets using RNeasy Mini Kit with on-column DNase digestion (Qiagen; Hilden, Germany). cDNA synthesis was performed as described in the Expression Analysis Technical Manual (Affymetrix; Santa Clara, CA). GAPDH was used as control gene and related gene expression (Bcl-2, Mcl-1, Bcl-xL, BID, and BAX), (all from IDT, Coralville, Iowa) was analyzed on an Applied Biosystems 7500 Real-Time PCR System using Taqman Universal PCR Master Mix #4304437 and Assay on Demand primer/probe kits (Applied Biosystems; Waltham, MA). For TLDA assay, TLDA v2.0 was performed on the 7900HT real-time PCR system (Applied Biosystems) according to the manufacturer’s protocol. Average delta CT was acquired from the results for further analysis. PCR cycling conditions were performed as follows: 95 °C for 15 s and 60 °C for 1 min, 40 cycles and then 95 °C for 10 min. To normalize RNA input, Human RNU44 small RNA was used as an internal control.