TABLE 1.
Real-time reverse transcriptase-PCR quantification of virus and cell RNAsa
| RNA source | 7SLb | Actinb | 7SL/actinc |
|---|---|---|---|
| aDNA was removed from samples using DNase I (Ambion). First-strand cDNA was synthesized using random primers and SuperScript II (Invitrogen). After RNaseH treatment, samples were diluted in herring DNA and PCR was performed by TaqMan using an ABI 7700 system (Perkin Elmer). Primers for 7SL: 5′-GGGCTGTAGTGCGCTATGC-3′ and 5′-CCCGGGAGGT CACCATATT-3′; probe: 6FAM-CGGGTGTCCGCACTAAGTTCGGCTAMRA. Primers for beta-actin: 5′-TCACCCACACTGTGCCCATCTACGA-3′, and 5′-CAGCGGAACCGCTCATT GCCAATGG-3′; probe: 6FAM-ATGCCCTCCCCCATGCCATCCTGCGT-TAMRA. Standard curves were generated using dilutions of plasmid target. Calculations were based on the linear response of the threshold cycle when PCR products were first detected versus log input template copy number. Linear response ranged from 10 to 106 template copies, achieving correlation coefficients of >0.99. | |||
| bAmounts of 7SL or actin sequences present in dilutions of cDNAs produced from cellular or viral RNA by the addition of random primers plus reverse transcriptase, as determined by quantitative PCR. Standard errors are indicated with ±. | |||
| cMolar ratios of 7SL to actin were calculated from presented 7SL and actin data. Values from controls in which reverse transcriptase was not included were subtracted from presented values; these -RT values were 12 ± 5.0 template copies for CEM-SS cells’ 7SL, 6.7 for CEM-SS’ actin, 160 ± 21 for infected cells’ 7SL, 24 ± 3.5 for infected cells’ actin, 1.5 ± 0.3 for virions’ 7SL, and 9.1 ± 4.1 for virions’ actin. | |||
| CEM-SS cells | 5.2 ± 0.2 × 107 | 7.2 ± 0.1 × 106 | 7.2 ± 0.9 × 103 |
| Infected CEM-SS cells | 1.3 ± 0.1 × 108 | 4.2 ± 0.9 × 106 | 3.1 ± 0.7 × 101 |
| HIV-1BRU virions | 6.2 ± 0.3 × 107 | 7.8 ± 1.9 × 102 | 8.0 ± 2.0 × 103 |
