Article Figures & Data
Figures
- FIG 1
Agilent TapeStation electropherogram, showing a representative plot of DNA from the U1 cell line before and after PFGE. From 500 bp to 15 kb, statistical analysis by the Mann-Whitney unpaired nonparametric t test determined a significant difference for results before versus after PFGE (P = 0.0079).
- FIG 2
HIV sequence copies per million cells using ddPCR with DNA from PBMCs from 9 subjects not receiving ART, before or after PFGE. HIV gag was measured to capture both proviral DNA and HIV episomes. 2-LTR primers were used to measure 2-LTR circular episomes. (a) HIV gag and 2-LTR circle copy numbers for the individual subjects. (b) Median values for the 9 subjects. The median gag copy number before PFGE was 2,227 copies per million cells (95% CI, 1,006 to 2,769 copies per million cells). The median gag copy number after PFGE was 542 copies per million cells (95% CI, 210 to 1,584 copies per million cells) (P = 0.0078). The median 2-LTR circle copy number before PFGE was 22 copies per million cells (95% CI, 0.76 to 120 copies per million cells); after PFGE, no 2-LTR circles were detected in any of the samples (P = 0.0078). The median recovery of genomic DNA of 37% (95% CI, 28.9 to 42.8%) was determined by measuring the host cell RPP30 gene.
- FIG 3
(a and c) HIV gag DNA copies per million cells before PFGE for complete technical replicates of PBMCs from 11 ART-suppressed subjects. The median for the first replicate (A) was 122.3 copies per million cells (95% CI, 62.6 to 335.5 copies per million cells), and that for the second replicate (B) was 120.9 copies per million cells (95% CI, 59.95 to 388 copies per million cells). By Wilcoxon's paired nonparametric t test, no significant difference was observed between replicates. (b and d) HIV gag DNA copies per million cells after PFGE for complete technical replicates of cells from 11 ART-suppressed subjects. The median for the first replicate (A) was 93 copies per million cells (95% CI, 36.4 to 160.2 copies per million cells), and that for the second replicate (B) was 97.8 copies per million cells (95% CI, 52.7 to 195.3 copies per million cells). By Wilcoxon's paired nonparametric t test, no significant difference was observed between replicates. (e) HIV gag DNA copies per million cells before or after PFGE for PBMCs from 22 ART-suppressed subjects. The median gag copy number before PFGE was 270.4 copies per million cells (95% CI, 193.7 to 486.6 copies per million cells), and that after PFGE was 160.6 copies per million cells (95% CI, 89.6 to 399.5 copies per million cells). The median genomic DNA recovery percentage was 22.5% (95% CI, 20.4 to 29.6%), as determined with the RPP30 gene. Wilcoxon's paired nonparametric t test determined a significant difference for results before versus after PFGE (P = 0.0017).
- FIG 4
(a and b) Correlations between values obtained using Alu-gag qPCR and HIV gag ddPCR with samples from 10 ART-suppressed subjects, normalized to copies per million cells, before (a) and after (b) PFGE. The HIV gag PFGE and ddPCR were performed on the DNA with blinding to the results of the Alu-gag qPCR. The r and P values were determined using Spearman's rho correlations. (c) Values in panels a and b plotted together, to provide visual comparison. (d) Comparison of integrated HIV DNA copies per million cells determined using Alu-gag qPCR and gag ddPCR after PFGE, with DNA from PBMCs collected from 10 ART-suppressed subjects. Wilcoxon's paired nonparametric t test determined no significant difference between the methods.
