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Table 1 Assays to measure CA HIV RNA in HIV-infected individuals

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

[85, 119, 121]

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

[4, 6, 122]

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

[26, 65]

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

[78, 130, 131]

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

[48, 141]

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

[40, 142, 143]