Quantitative PCR for Plasma Viral Load

Plasma viral load was measured by quantitative real-time PCR (qPCR)64 (link). RNA was extracted from 50 μl plasma using the Qiagen RNAeasy kit and eluted in 100 μl water. RNA (10 μl) was added to 1 × 1-step TaqMan Reverse Transcriptase PCR reaction mix (Applied Biosystems) along with 10 pmol of the following primers and probe: F-MuLV sense, 5′-GGACAGAAACTACCGCCCTG-3′; F-MuLV antisense, 5′-ACAA- CCTCAGACAACGAAGTAAGA-3′; and F-MuLV probe, FAM-TCGCCACCCAGCAGTTTCAGCAGC-TAMRA. Real-time PCR was performed in a Bio-Rad CFX96 cycler using these thermocycling conditions: 48 °C for 15 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min. T7-transcribed F-MuLV RNA standards were used to interpolate absolute F-MuLV RNA copy numbers in the plasma.