Quantifying Intact Proviral DNA by ddPCR

The intact proviral DNA assay was performed as described by Bender et al. (47 (link)), including thermal cycling conditions, primers, and probes used in this assay. In brief, 250 to 1,000 ng of DNA was mixed with 10 µl of 2× ddPCR Supermix for probes (no dUTP) (Bio-Rad; cat. no. 863024), 600 nM primers, and 200 nM probes. ddPCR was carried out on a Bio-Rad QX200 AutoDG digital droplet PCR system as described above. To quantify the input cell numbers, a parallel ddPCR reaction for the rhesus macaque RPP30 gene was carried out.

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Acharya A., Olwenyi O.A., Thurman M., Pandey K., Morsey B.M., Lamberty B., Ferguson N., Callen S., Fang Q., Buch S.J., Fox H.S, & Byrareddy S.N. (2021). Chronic Morphine Administration Differentially Modulates Viral Reservoirs in a Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaque Model. Journal of Virology, 95(5), e01657-20.

Publication 2020

QX200 AutoDG digital droplet PCR system

A gene Assay Dutp Primers Proviral Rhesus macaque

Corresponding Organization : University of Nebraska Medical Center

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Variable analysis

independent variables

Thermal cycling conditions
Primers
Probes

dependent variables

Quantification of intact proviral DNA

control variables

Amount of DNA (250 to 1,000 ng)
Concentration of primers (600 nM)
Concentration of probes (200 nM)
DdPCR reaction for the rhesus macaque RPP30 gene to quantify input cell numbers

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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