We measured the HIV proviral reservoir in genomic DNA from CD4+ T cells and from central memory, effector memory, and naïve T-cell subsets. We also measured HIV in rectal biopsies. Methods used for isolating and sorting T-cell subsets are described in the Supplementary Methods . We used the Bio-Rad ddPCR platform to run an assay targeting 3 regions in the HIV genome (within gag, pol, and env). This assay provides a good approximation of intact and defective provirus copy numbers, has been validated in a clinical laboratory, and is described in detail in 2 prior publications [31 (link), 32 (link)]. A determination of proviral “intactness” by this method requires the detection of all 3 HIV targets in a single droplet. To avoid false negatives from true intact provirus being mechanically sheared and distributed into different droplets, we used a method modified from Wiegand et al. to extract high–molecular weight genomic DNA [33 (link)]. For rectal biopsies, we used the adapted Wiegand protocol and a Qiagen QIAamp DNA micro kit. Additional details are available in the Supplementary Methods .
Ddpcr platform
Manufactured by Bio-Rad
The DdPCR platform is a digital PCR (polymerase chain reaction) system developed by Bio-Rad. It is designed to provide precise and sensitive quantification of nucleic acid targets. The platform partitions samples into thousands of individual droplets, allowing for the simultaneous amplification and detection of target sequences within each droplet. This approach enables accurate quantification of target molecules, even at low concentrations.
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5 protocols using ddpcr platform
1
Measuring HIV Proviral Reservoir in T Cell Subsets
2
Calculating Cell-Free DNA Mutant Burden
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To calculate mutant copies per mL of plasma in each timepoint, the mutant copies per microliter (as obtained from the ddPCR platform (Bio-Rad)) were transformed into mutant copies per eluate:
Mutant copies per mL of plasma were calculated as follows:
For each timepoint, ctDNA VAF was transformed into MGE as follows:
Mutant copies per mL of plasma were calculated as follows:
For each timepoint, ctDNA VAF was transformed into MGE as follows:
3
Prostate CTC ddPCR Assay Development
4
Synthetic SIV gag qPCR Assay
5
Absolute Quantitation of miRNAs using ddPCR
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For absolute quantitation of miRNAs, we used a ddPCR platform (Bio Rad Inc.). For ddPCR reaction, 10 ng of total RNA was reverse transcribed using the specific miRNA TaqMan assays (Thermofisher Scientific Inc.) as per recommended protocol in a 15 μL total reaction volume; 5 μL of reverse-transcribed product was used to set up the real-time PCR reaction using miRNA TaqMan assays; and 20 μL of the final real-time PCR reaction was mixed with 70 μL of droplet oil in a droplet generator (Bio Rad Inc.). Following the droplet formation, the PCR reaction was performed as per recommended thermal cycling conditions. The final PCR product within the droplets was analyzed in a droplet reader (Bio-Rad Inc.). The total positive and negative droplets were measured, and the concentration of the specific miRNA/μL of the PCR reaction was determined. All the reactions were performed in duplicates.
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