Figure 6

PCR-Quantification of HIV-RNAs. A: Location of primers and probes used for PCR. Labeled bars depict viral genes and the unlabelled bars below show exons of viral transcripts quantified as previously described [15]. White: genomic RNA spanning the entire transcriptional unit, grey: multiply spliced RNAs encoding tat and rev (dark grey) or nef (light grey). Arrows show the location of PCR primers and barbells the positioning of fluorescent probes. Note that MsRNA-nef was calculated as the difference of copy number for MsRNA-total and MsRNA-tatrev as described previously [15] (formula indicated in italic letters). B: Performance of PCR assays. Patient-specific in vitro transcribed UsRNA and MsRNA prepared from templates isolated from patient 104 and quantified photometrically was subjected to RT-PCR using patient specific primers as listed in table 3. Ten-fold dilutions ranging from 5 × 10⁷ – 5 copies were run in replicates of two (5 × 10⁷ – 5 × 10⁴ copies) or four (5000–5 copies). Upper panels show amplification plots (mean ± standard error) with the amplification threshold (broken lines) as calculated by default settings of the software used (i-cycler, Biorad). In the lower panels log-transformed copy numbers were plotted against cycle-threshold (ct) values of each replicate. Linear regression analysis as shown by the resulting equations for black lines (broken lines depict 95% confidence intervals) revealed high correlation coefficients of standard curves. PCR efficiencies as deduced from slopes ranged from 89%–96%. Sensitivities of PCR assays (grey vertical lines, italic numbers) were estimated by determination of 50% endpoints [51]. All assays approached single copy sensitivities as previously documented [15, 22] with assays for MsRNA-tatrev (1.6 copies) being the most sensitive ones followed by PCR for UsRNA/vRNA-ex (2.3 copies) and the assay for MsRNA-total (5 copies). PCR assays for the other patients performed similarly (data not shown).