Quantitative RT-PCR (qRT-PCR).

Total RNA was isolated from 10⁶ cells per condition using an RNeasy minikit (Qiagen) according to the manufacturer's instructions. Isolated RNA was quantitated and DNase treated using recombinant DNase I (Roche). Reverse transcription (RT) was performed using a SuperScript first-strand synthesis system for RT-PCR (Life Technologies) according to the manufacturer's instructions. The instrumentation and general principles of the CFX96 Touch real-time PCR detection system (Bio-Rad) are described in detail in the operator's manual. PCR amplification was carried out in 96-well plates with optical caps. The final reaction volume was 20 μl and consisted of 10 μl iQ SYBR green Supermix (Bio-Rad), 300 nM each specific primer, and 2 μl of cDNA template. For each run, standard cDNA, sample cDNA, and a no-template control were all assayed in triplicate. The reaction conditions were 95°C for 5 min, followed by 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s. Primer pairs for the specific detection of viral mRNA species (gag/pol, tax/rex, hbz, aph-2), platelet-derived growth factor beta polypeptide (pdgfb), and human glyceraldehyde-3-phosphate dehydrogenase (hgapdh) were described previously (45,–47). Data from triplicate experiments are presented in histogram form as means with standard deviations. The total copy number for each viral gene was determined using a plasmid DNA standard curve and normalized to 10⁶ copies of hGAPDH mRNA.