qPCR analysis of miRNA expression.

Total RNAs, including small RNA from transfected, lentivirus-transduced, or HIV-infected HeLa-CD4/CXCR4/CCR5 cells, primary MDMs, and lymphocytes, were purified using the miRNeasy minikit (Qiagen) according to the manufacturer’s instructions. Mature miRNAs and certain small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) were selectively reverse transcribed into cDNA using miScript HiSpec buffer according to the instructions of the miScript II RT kit (Qiagen). Briefly, mature miRNAs were polyadenylated by poly(A) polymerase and reverse transcribed into cDNA using oligo(dT) primers. Polyadenylation and reverse transcription were performed in parallel in the same reaction. The oligo(dT) primers included a 3′ degenerate anchor and a universal tag sequence on the 5′ end, allowing the amplification of mature miRNA in the real-time PCR step.

The resulting cDNAs served as the templates for real-time PCR analysis using miRNA-specific forward primers (IDT) and the miScript SYBR green PCR kit (Qiagen), which contains the miScript universal primer (reverse primer) and QuantiTect SYBR green PCR master mix. The amplification cycles consisted of an initial activation step at 95°C for 15 min, followed by 40 cycles of 15 s at 94°C, 30 s at 55°C, and 30 s at 70°C. Fluorescence data were collected during the 70°C extension step. The miRNA targets and primers that were used in this study were previously described (11). As an internal control, the levels of a small nuclear RNA, RNU6B (a miScript PCR control provided in the miScript PCR starter kit [Qiagen]), were determined. Relative miRNA expression was normalized to RNU6B levels using the comparative threshold cycle (ΔΔC_T) method. All miRNA expression studies were conducted using an Mx3005P thermocycler (Stratagene, La Jolla, CA).