From: What do we measure when we measure cell-associated HIV RNA
Bulk or single-cell? | Chemistry | Assay type | Pros | Cons | References |
---|---|---|---|---|---|
Bulk | qPCR | RT-qPCR | Sensitive, high throughput, low cost | Depends on external standards, no single-cell analysis possible, sensitive to primer/probe-template mismatches | |
RT-seminested qPCR | Highly sensitive, high throughput, increased accuracy in the lower quantitation range, low cost | Depends on external standards, no single-cell analysis possible, sensitive to primer/probe-template mismatches | |||
Digital PCR | RT-ddPCR | Sensitive, high throughput, no standard curve needed, less affected by primer/probe-template mismatches | False positive droplets, possible underestimation due to molecular dropout, external calibrator needed to account for the yield of the RT reaction, higher cost compared to qPCR, no single-cell analysis possible | ||
Single-cell | PCR | Limiting dilution-RT-qPCR or -RT-ddPCR | Estimates frequencies of CA RNA+ cells | Costly, laborious, in its current form does not report CA RNA copy numbers per cell | |
Limiting dilution-RT-PCR-sequence analysis | Estimates frequencies of CA RNA+ cells and CA RNA copy numbers per cell, allows to estimate intactness/defectiveness and clonal expansion of expressed proviruses | Costly, laborious, does not directly measure frequencies of CA RNA+ cells and CA RNA copy numbers per cell, only 15% of HIV genome sequenced so mismatches outside this region cannot be accounted for | [133] | ||
Single-cell-in-droplet-ddPCR | High-throughput, allows direct quantitation of CA RNA+ cells, lower cost compared to other single-cell methods | In its current form does not report CA RNA copy numbers per cell, possible underestimation of CA RNA+ cell numbers due to “cellular dropout” | [135] | ||
In situ hybridization | Microscopy-based | Allows direct visualization and characterization of CA RNA+ cells, duplex HIV RNA and DNA detection in the same tissue section possible, low background | Does not report CA RNA copy numbers per cell, possibly reduced sensitivity compared to PCR-based assays, laborious, relatively low throughput | ||
Flow-based | Allows characterization of CA RNA+ cells, can be combined with HIV protein detection | Does not report CA RNA copy numbers per cell, reduced sensitivity compared to PCR-based assays, high background |