(A) RT-PCR analysis of the effect of IFN-γ on CCR5 mRNA. Monocytes were incubated with IFN-γ (500 U/mL) and total cellular RNA was extracted at 30 minutes, 1 hour, 2 hours, and 4 hours after incubation with IFN-γ. (B) Competitive PCR analysis for quantification of mRNA. cDNA from the samples (untreated monocytes and monocytes incubated with IFN-γ for 30 minutes) were mixed with varying dilutions of the competitor DNA fragment and amplified with CCR5 primer sets. Lanes 1 through 6 represent increasing dilutions of competitor DNA fragment (3 × 106, 1.5 × 106, 0.7 × 106, 0.35 × 106, 3 × 105, and 3 × 104 molecules/μL). Amplification products were resolved by 4% agarose gel electrophoresis and the ratios of competitor/CCR5 DNA for untreated and IFN-γ–treated cells were determined by densitometric scanning. Lane to lane variation in RNA loading was less than 18% as assessed by densitometric analysis of β-actin expression. Representative results from 4 cord blood samples are shown.
