Stat 60

As previously described, CCRF-CEM cells were infected with HIV-1 IIIB for 5 days (MOI 0.001) and then treated twice with experimental RNAs at 800 nM final concentration in 12-well plates at day 1 and day 3. After 7 days of incubation, total RNA was isolated with STAT60 (TEL-TEST, Friendswood, TX, USA) according to the manufacturer's instructions. Residual DNA was digested using the DNA-free kit per the manufacturer's instructions (Ambion, CA, USA). cDNA was produced using 2 µg of total RNA. Reverse transcription was carried out using Moloney murine leukaemia virus reverse transcriptase (MMLV-RT) and random primers in a 15 µL reaction according to the manufacturer's instructions (Invitrogen, CA, USA). Expression of the TNPO3 coding RNAs was analyzed by quantitative RT-PCR using 2× iQ SyberGreen Mastermix (Bio-Rad) and specific primer sets at a final concentration of 400 nM. Gapdh expression was used for normalization of the qPCR data. Primers were as follows: HIV-1 IIIB tat/rev forward primer: 5'-GGC GTT ACT CGA CAG AGG AG-3'; HIV-1 IIIB tat/rev reverse primer: 5'-TGC TTT GAT AGA GAA GCT TGA TG-3'. HIV-1 Pol p128 forward primer: 5'-AGG GAT GGA AAG GAT CAC CAG CAA-3'; HIV-1 Pol p129 HIV reverse primer: 5'-CCC ACC TCA ACA GAT GTT GTC TCA-3'. GAPDH forward primer: 5'-CAT TGA CCT CAA CTA CAT G-3'; GAPDH reverse primer: 5'-TCT CCA TGG TGG TGA AGA C-3'.