Table 1 Characteristics of assays measuring the inducible HIV reservoir
From: Inducible HIV RNA transcription assays to measure HIV persistence: pros and cons of a compromise
References | Formata and applicationb | Target transcript | Stimulation method | Stimulation time (hours) | RT-ddPCR/RT-qPCR/scdPCRc | Required CD4 T cells/assay (million) | Advantages | Limitations | |
|---|---|---|---|---|---|---|---|---|---|
cfRNA | caRNA | ||||||||
Procopio et al. [14] | LD RQ + LRA | – | tat/Rev | PMA + ionomycin | 12 | RT-qPCR | < 1 | Fast, simple, robust and sensitive method, with no RNA extraction and small amounts of cells required Measures frequency of cells expressing msRNA | Does not evaluate RNA levels produced in individual cell |
Yukl et al. [11] | B LRA | – | Profiling panel | aCD3/CD28 + IL-2 | 24–48 | RT-ddPCR | 6–10 | Exhaustive analysis of the modulation of the transcriptional profile upon activation Enables the identification of blocks HIV transcription, elongation and splicing after stimulation | Does not evaluate RNA levels produced in individual cell |
Bullen et al. [17] | B LRA | poly-A | Profiling panel | PMA + ionomycin or LRA | 6–18 | RT-qPCR | 5–10 | Analysis of the modulation of the transcriptional profile upon activation Enables the quantification of mature HIV transcripts Adapted to evaluate the activity of LRAs | Does not evaluate RNA levels produced in individual cell |
Massanella et al. [13] | LD RQ + LRA | gag | gag tat/rev | aCD3/CD28 | 72 | RT-ddPCR | 4.5–9 | Combines the value of frequency based assays with cell-associated/cell-free assays Great potential for translational analysis from provirus to virion detection Measures frequency of cells expressing msRNA | Labour intensive Does not evaluate RNA levels produced in individual cell |
Cillo et al. [10] | LD RQ + LRA | pol | pol | aCD3/CD28or SAHA | 168 | RT-qPCR | 12 | Combines the value of frequency based assays with cell-associated/cell-free assays Good potential for translational analysis from provirus to virion detection | pol RNA increases chances of measuring defective proviruses Labour intensive Does not evaluate RNA levels produced in individual cell |
Fromentin et al. [15] | B RQ + LRA | LTR-gag | – | aCD3/CD28 or LRA | 168 | RT-qPCR | 5 | Fast and simple Adapted to evaluate the activity of LRAs | Does not evaluate RNA levels produced in individual cell |
Yucha et al. [12] | B + SC RQ + LRA | – | gag tat/rev | aCD3/CD28 or LRA | 18 | scdPCR/RT-ddPCR/RT-qPCR | 0.5–1 | Allows characterization and quantification of transcriptionally active cells in response to various stimuli High-throughput and reduced costs compared to other single-cell platforms Can be adapted to other HIV transcripts Enables the characterization of reservoir cells by rescuing genomic and viral DNA as well as mRNA | Technically demanding |