May 29, 2026
ddPCR Protocol: Detection and Quantification of Integrated HIV Psi Sequence
- Hayley Todesco1,
- Rebecca Malott1,
- Kyle Potts1,
- Douglas Mahoney1
- 1University of Calgary
Protocol Citation: Hayley Todesco, Rebecca Malott, Kyle Potts, Douglas Mahoney 2026. ddPCR Protocol: Detection and Quantification of Integrated HIV Psi Sequence. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6ok9klqe/v1
Manuscript citation:
Zemp et al. 2026. GPNMB-directed CAR T-cell therapy against MiT/TFE family fusion-driven solid tumors. Nature Cancer. Accepted.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 27, 2026
Last Modified: May 29, 2026
Protocol Integer ID: 318072
Keywords: ddPCR, Vector Copy Number, Droplet Digital PCR, QX600, quantification of integrated hiv psi sequence detection, integrated hiv psi sequence detection, droplet digital pcr, ddpcr protocol, ddpcr, reference gene rpp30, targeting psi, following lentiviral, rad qx600
Abstract
Detection and quantification of the HIV-1 Psi sequence integrated into cells following lentiviral (LV) transduction. Performed as singleplex reactions for targeting Psi or the reference gene RPP30 using the Bio-Rad QX600™ Droplet Digital PCR (ddPCR)™ system with AutoDG following manufacturer instructions.
Materials
- Phosphate-Buffered Saline, pH 7.4
- DNeasy Blood 26 Tissue Kit (Qiagen, cat. no. 69504)
- RNase A (Qiagen, cat. no. 19101)
- Anhydrous ethanol, 3e96%
- Qubit 1X dsDNA HS assay kit (Thermo Scientific, cat. no. Q33230)
- Qubit assay tubes (Thermo Scientific, cat. no. Q32856)
- Nuclease-free water
- ddPCR 96-well plates (Bio-Rad, cat. no. 12001925)
- 2× ddPCR Supermix for Probes (no dUTP; Bio-Rad, cat. no. 1863024)
- BamHI-HF restriction enzyme (New England Biolabs, cat. no. R3136L)
- rCutSmart™ Buffer (New England Biolabs, cat. no. B6004S)
- Custom primer/probe reagents:
- 20× HIV Psi sequence (FAM-labeled; Bio-Rad, dEXD14812826)
- 20× RPP30 reference gene (HEX-labeled; Bio-Rad, dHsaMDS117591774)
- Pierceable foil heat seals (Bio-Rad, cat. no. 17005225)
- DG32 AutoDG cartridges (Bio-Rad, cat. no. 1864108)
- Droplet generation oil for probes (Bio-Rad, cat. no. 1864110)
- Pipet tips for the AutoDG system (Bio-Rad, cat. no. 1864120)
- ddPCR droplet reader oil (Bio-Rad, cat. no. 1863004)
- Vortex (Fisher Scientific)
- Microcentrifuge (Fisher Scientific)
- Thermal mixer (Thermo Scientific)
- Qubit 4 Fluorometer (Thermo Scientific)
- Sorvall X4R PRO refrigerated centrifuge with TX-1000 rotor (Thermo Scientific)
- Bio-Rad QX600™ Droplet Digital PCR system coupled with AutoDG
- PX1 PCR Plate Sealer (Bio-Rad)
- PTC Tempo Deepwell Thermal Cycler (Bio-Rad)
- QX Manager Premium Edition Software (v2.3; Bio-Rad)
Protocol materials
Invitrogen™ UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10-977-015
PBS, pH 7.4Gibco - Thermo Fisher ScientificCatalog #10010023
Qubit 1X dsDNA HSThermo Fisher ScientificCatalog #Q33230 or Q33231
Qubit assay tubesThermo Fisher ScientificCatalog #Q32856
BamHI-HF - 50,000 unitsNew England BiolabsCatalog #R3136L
20× RPP30 Custom Probe/Primer ddPCR Assay, HEXBio-Rad LaboratoriesCatalog #dHsaMDS117591774
ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
rCutSmart BufferNew England BiolabsCatalog #B6004
20× HIV Psi Custom Probe/Primer ddPCR Assay, FAMBio-Rad LaboratoriesCatalog #dEXD14812826
ddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925
DNeasy Blood & Tissue Kit (QIAGEN)QiagenCatalog #69504
RNase AQiagenCatalog #19101
ddPCR™ Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004
DG32™ Automated Droplet Generator CartridgesBio-Rad LaboratoriesCatalog #1864108
Pipet Tips for the AutoDGBio-Rad LaboratoriesCatalog #1864120
Pierceable Foil Heat SealsBio-Rad LaboratoriesCatalog #17005225
Automated Droplet Generation Oil for ProbesBio-Rad LaboratoriesCatalog #1864110
Procedure
7h 50m
Genomic DNA Extraction:
Pellet 5*10^5 cells from isolated PBMCs at 300 x g, 4°C, 00:05:00 and remove the supernatant.
Equipment
Accuspin 21R Refrigerated Microcentrifuge
NAME
Centrifuge
TYPE
Fisherbrand
BRAND
13100677
SKU
10m
Resuspend cells in 200 µL of PBS, pH 7.4Gibco - Thermo Fisher ScientificCatalog #10010023 .
5m
Extract genomic DNA using the DNeasy Blood & Tissue Kit (QIAGEN)QiagenCatalog #69504 and >96% anhydrous ethanol. Follow the kit manufacturer’s instructions with an additional step of adding 4 µL RNase AQiagenCatalog #19101 per sample prior to the addition of Buffer AL.
1h
DNA Extract Dilution:
Quantify the dsDNA concentration of each DNA extract using the Qubit 1X dsDNA HSThermo Fisher ScientificCatalog #Q33230 or Q33231 and Qubit assay tubesThermo Fisher ScientificCatalog #Q32856 according to the manufacturer’s instructions.
Equipment
Qubit 4
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
10m
Prepare DNA extract dilutions containing ≤4.1 ng/uL (i.e. ≤33 ng per every 8 µL) in Invitrogen™ UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10-977-015 .
10m
ddPCR Reaction Setup (Singleplex):
Prepare each singleplex ddPCR reaction in a total volume of 22 µL in ddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925 in a laminar flow PCR workstation:
- ≤33 ng genomic DNA in 8 µL
- 1× ddPCR Supermix for probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863024
- 1 µL containing 5 units of BamHI-HF - 50,000 unitsNew England BiolabsCatalog #R3136L diluted in Invitrogen™ UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10-977-015 and 1× rCutSmart BufferNew England BiolabsCatalog #B6004
- 1× custom primer/probe reagent - either 20× HIV Psi Custom Probe/Primer ddPCR Assay, FAMBio-Rad LaboratoriesCatalog #dEXD14812826 or 20× RPP30 Custom Probe/Primer ddPCR Assay, HEXBio-Rad LaboratoriesCatalog #dHsaMDS117591774
1h
Droplet Generation:
Generate droplets using DG32™ Automated Droplet Generator CartridgesBio-Rad LaboratoriesCatalog #1864108 , Pipet Tips for the AutoDGBio-Rad LaboratoriesCatalog #1864120 , Pierceable Foil Heat SealsBio-Rad LaboratoriesCatalog #17005225 , and Automated Droplet Generation Oil for ProbesBio-Rad LaboratoriesCatalog #1864110 following the manufacturer’s instructions.
Equipment
PX1 PCR Plate Sealer
NAME
Plate Sealer
TYPE
Bio-Rad
BRAND
1814000
SKU
LINK
Equipment
Automated Droplet Generator
NAME
Droplet Digital PCR System
TYPE
Bio-Rad
BRAND
1864101
SKU
LINK
Equipment
Sorvall Legend X1R
NAME
Centrifuge
TYPE
ThermoScientific
BRAND
35m
Thermal Cycling:
Perform thermal cycling under the following conditions (heated lid set to 105 °C , maximum ramp rate of 2 °C per second):
- Initial enzyme activation: 95 °C for 00:10:00
- 40 cycles of: denaturation: 94 °C for 00:00:30 and annealing/extension: 55 °C for 00:01:00
- Enzyme deactivation: 98 °C for 00:10:00
- Hold: 4 °C for at least 00:05:00
Equipment
PTC Tempo Deepwell Thermal Cycler
NAME
Thermal Cycler
TYPE
Bio-Rad Laboratories
BRAND
12015392
SKU
2h
Droplet Reading:
Measure fluorescence intensity from a minimum of 10000 droplets per sample using ddPCR™ Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004 and the “Direct Quantification” setting on all loaded wells.
Equipment
QX600 Droplet Reader
NAME
ddPCR Instrument
TYPE
Bio-Rad Laboratories
BRAND
12013328
SKU
Software
QX Manager Premium Edition
NAME
Bio-Rad Laboratories
DEVELOPER
2h
Data Analysis:
Using 1D amplitude plots, apply manual amplitude thresholds:
- 1000 amplitude units for FAM (Psi target)
- 2000 amplitude units for HEX (RPP30 reference)
Software
QX Manager Premium Edition
NAME
Bio-Rad Laboratories
DEVELOPER
30m
Use the threshold-adjusted concentrations calculated by the software to calculate the Psi:Cell value using the following formula:
Psi:Cell = (Psi concentration (copies/µL)) / (0.5 × RPP30 concentration (copies/µL))
10m
Notes
All steps involving the ddPCR system and AutoDG must follow the manufacturer’s instructions.
Run Psi (target) and RPP30 (reference) reactions in triplicate singleplex wells.
Ensure accurate DNA quantification prior to reaction setup for reliable copy-number calculations.
Include appropriate controls including DNA extraction controls and no-template controls.
Acknowledgements
Notes
- All steps involving the ddPCR system and AutoDG must follow the manufacturer’s instructions.
- Run Psi (target) and RPP30 (reference) reactions in triplicate singleplex wells.
- Ensure accurate DNA quantification prior to reaction setup for reliable copy-number calculations.
- Include appropriate controls including DNA extraction controls and no-template controls.