Table 1 Comparison of single-cell approaches to measure the transcription- and translation-competent reservoirs
From: Beyond the replication-competent HIV reservoir: transcription and translation-competent reservoirs
Cellular reservoir measured | Assay | Assay overview | Advantages | Limitations | Potential applications | Key references |
|---|---|---|---|---|---|---|
Transcription-competent cellular reservoir | RNAflow cytometry | Detection of cells expressing HIV RNA in suspension by fluorescence in situ hybridisation (FISH) using branched DNA (bDNA) technology | High throughput In depth phenotyping of single cells Highly flexible and adaptable | Background observed in HIV-uninfected individuals Labour intensive (2–3 days protocol) High starting cell number required | LRA screening Reservoir quantification In depth phenotyping of the reservoir Biomarker discovery | |
Simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI) | Detection of cells expressing HIV RNA in suspension by fluorescence in situ hybridisation (FISH) | High throughput | Higher than predicted frequencies of mRNA+ cells observed | Reservoir quantification Phenotyping of single cells, including myeloid cells | ||
Conventional in situ hybridization | Detection of cells expressing HIV RNA in situ using radiolabelled or enzymatic detection | Tissue level information | Limited cell phenotyping Labour intensive | Reservoir quantification in tissues | ||
RNAScope | Detection of cells expressing HIV RNA in situ using branched DNA amplification and detection | Tissue level information Highly sensitive and specific Short assay duration | Limited cell phenotyping | Reservoir quantification in tissues/whole body | ||
Translation-competent cellular reservoir | Fiber optic array scanning technology (FAST) | Antibody-based detection of cells expressing HIV protein and down-regulating CD4, in suspension | Relatively high throughput Short assay duration | Specialized microscopy tools and software required Limited cell phenotyping | LRA screening Reservoir quantification | [62] |
RNAflow cytometry | Concurrent detection of cells expressing HIV RNA by FISH using branched DNA (bDNA) technology, and HIV protein in suspension | High linearity and specificity High throughput In depth phenotyping of single cells Highly flexible and adaptable | Labour intensive (2–3 days protocol) High starting cell number required | LRA screening Reservoir quantification In depth phenotyping of the reservoir Biomarker discovery |