Real-time PCR for antiretroviral interferon-stimulated genes (ISGs).

RNA was isolated from 5 × 10⁶ splenocytes using the ZR-Duet DNA/RNA MiniPrep kit (Zymo Research, Irvine, CA). Real-time PCR analysis of mRNA expression was completed as previously described (50). Primers were tested on controls to ensure amplification of a single product. Data for each sample were calculated as the percent difference in the threshold cycle (C_T) value (ΔC_T = C_T GAPDH − C_T gene). Gene expression was plotted as a percentage of gene expression relative to that of GAPDH for each sample.