Fig. 1.
RT-PCR detection of RNAs packaged within native HIV-1 particles. Total RNA was isolated from pelleted and purified native HIV-1 infectious particles, where viral particles (equivalent of ~650 ng p24) were also treated with RNase in the presence or absence of Triton X-100 for 1 h at 37 ° C in a total volume of 100 μL prior to RNA extractions as indicated. Total RNA was then reverse-transcribed using random hexamer primers to generate cDNA templates used for RT-PCR detection of the indicated RNAs, which included HIV-1 genomic RNA (gRNA), fully-spliced viral mRNA (Tat) and cellular small RNAs and mRNAs. Primer sequences and RT-PCR product sizes are listed in Table 1. RT-PCR products were detected using 2% agarose gels stained with ethidium bromide. For RT-PCR reaction controls, reverse transcription of DNase-treated RNA without the addition of reverse transcriptase was input to serve as a negative control (NTC, no cDNA template control), and HIV-1IIIB Tat1-86 full-length cDNA plasmid (pMax-Tat) and cDNA from primary human astrocytes were used to serve as positive controls for the amplification of Tat and cellular RNAs, respectively. Marker (M) indicates 100 base pair (bp) DNA ladder marker where the 100, 200 and 300 bp markers are shown.