Oct 31, 2025

Public workspaceDigital PCR Analysis for Mitochondrial DNA Integrity and Copy Number (Absolute Q MAP16 Format)

DOI
dx.doi.org/10.17504/protocols.io.dm6gpm1w5gzp/v1
  • Jenny Ghelfi1,
  • Soraya Hezzaz1,
  • Sylvie Delcambre1,
  • Anne Grünewald1,2
  • 1Luxembourg Centre for Systems Biology (LCSB), University of Luxembourg, Luxembourg;
  • 2Institute of Neurogenetics, University of Lübeck, Germany
jenny.ghelfi
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpm1w5gzp/v1
Protocol Citation: Jenny Ghelfi, Soraya Hezzaz, Sylvie Delcambre, Anne Grünewald 2025. Digital PCR Analysis for Mitochondrial DNA Integrity and Copy Number (Absolute Q MAP16 Format). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpm1w5gzp/v1
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: October 29, 2025
Last Modified: November 01, 2025
Protocol Integer ID: 231061
Keywords: dPCR, mitochondrial deletion, mitochondrial transcription, copy number, digital pcr analysis for mitochondrial dna integrity, digital pcr analysis, mitochondrial dna integrity, digital pcr, mitochondrial dna, setup of digital pcr, mtdna
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: 15744
Michael J. Fox Foundation
Grant ID: 025802
Michael J. Fox Foundation
Grant ID: 021147
Abstract
This protocol describes the preparation and setup of digital PCR assays to quantify mitochondrial DNA (mtDNA) integrity, deletion load, and copy number using the Absolute Q MAP16 format.

Materials
ABCD
ItemSupplierCatalog / Article No.Notes
QuantStudio™ Absolute Q™ MAP16 Plate Kit and Master MixThermo Fisher ScientificCat. #A53301Includes MAP16 plates and 5× Master Mix.
DLOOP-JUN/QSY ProbeThermo Fisher ScientificCustom, Article No. CCU002NRTarget: D-Loop region.
ND4-ABY/QSY ProbeThermo Fisher ScientificCustom, Article No. CCU002NRTarget: ND4 gene.
B2M-FAM/MGB ProbeThermo Fisher ScientificAssay ID APCFGXE, Cat. #43320Target: B2M (nuclear reference).
ND1-VIC/MGB ProbeThermo Fisher ScientificAssay ID APAANDG, Cat. #44485Target: ND1 gene.
Water, nuclease-freeThermo Fisher ScientificCat. #R0582For all dilutions and reactions.
TE Buffer, 1× (100 mL)PromegaCat. #V6231Used for probe dilution.
Ethanol (70%)VWR International (Avantor)930.031.006For surface cleaning.
ABCD
EquipmentBrandModel / SKULink / Notes
QuantStudio™ Absolute Q™ Digital PCR SystemThermo Fisher Scientific—Used for MAP16 plate runs.
Mini MicrocentrifugeCorningModel 6770Corning LSE Mini Microcentrifuge
Vortex MixerThermo ScientificCat. #88882011Basic Vortex Mixer
Protocol materials
Reagent70% ethanolVWR International (Avantor)
ReagentAbsolute Q™ DNA Digital PCR Master Mix (5X)Thermo Fisher ScientificCatalog #A52490
ReagentWater, nuclease-freeThermo FisherCatalog #R0582
Troubleshooting
Safety warnings
  • Wear appropriate personal protective equipment (PPE): laboratory coat, gloves, and safety glasses at all times.
  • Clean all work surfaces and equipment with 70% ethanol before and after use.
  • Dispose of biological waste, tips, and consumables according to your institution’s biosafety and chemical waste disposal procedures.
  • Follow the manufacturer’s safety data sheets (SDS) for all reagents and probes.
Before start
Samples should be extracted using a clean, preferably column-based method to minimize imaging artifacts during digital PCR. It is recommended to test a small range of concentrations before scaling up to a full plate.
Probe Preparation
10m
Check the format of probes:
  • Use 20× probes directly
  • If supplied as 60×, dilute to 20× in 1× TE buffer (20 µL probe + 40 µL TE)

Quencher guideline: 
A maximum of two MGB-quenched assays should be combined per reaction to ensure optimal fluorescence separation. QSY quenchers are compatible without limitation.

Digital PCR Setup (Absolute Q MAP16 Format)
40m
Plate Preparation
DNA template: Use DNA from CSF exosomes, PBMCs, or whole blood
  • Avoid impure extracts to prevent imaging artifacts
MAP16 plate capacity: 
Up to 16 samples
Unused columns may be sealed and stored for future runs
Prepare the master mix for each reaction:
  • Amount2 µL ReagentAbsolute Q™ DNA Digital PCR Master Mix (5X)Thermo Fisher ScientificCatalog #A52490 , vortex well before pipetting
  • Amount0.5 µL of each 20× probe mix (or Amount0.166 µL if using 60× probes)
  • Add ReagentWater, nuclease-freeThermo FisherCatalog #R0582 , variable volume (depending on DNA input later, final volume of master mix and DNA = Amount10 µL )
  • Prepare one extra reaction to account for pipetting loss
  • Vortex 
Combine DNA and master mix:
  • For CSF exosomes: Amount5 µL DNA +Amount5 µL master mix
  • For PBMC or whole blood DNA: Amount1.1 µL µL DNA + Amount8.9 µL master mix
Vortex and spin down to collect contents
Load the MAP16 plate:
  • Add Amount9 µL sample (DNA and master mix) to each well
  • Add Amount15 µL isolation buffer
  • Pipette at a 45° angle to avoid damaging the bottom of wells
  • Apply gasket strips after each loaded column
  • Seal unused columns
Running the Plate on the Absolute Q
1h 20m
Run preparation
Thermal Cycling Parameters
  • Pre-heat: 96 °C for 10 min
  • 40 cycles of:
  • 96 °C for 5 s
  • 60 °C for 30 s
Instrument Preparation and Run Start
  • Clean the instrument surface with Reagent70% ethanolVWR International (Avantor)  and allow to dry completely.
  • Load the prepared MAP16 plate into the Absolute Q instrument
  • Verify plate orientation matches the software layout
  • Start the run

Expected Results and Data Analysis
20m
Expected Results and Analysis
Expected Results
  • Clean partition images 
  • Clean separation of positive and negative partitions
  • Multiplex fluorophores:
  • VIC (ND1), FAM (B2M), ABY (ND4), JUN (D-loop).
Analysis
Quantitative results should be analyzed using the Absolute Q software or equivalent digital PCR analysis platform. Calculate the following ratios for interpretation of mitochondrial DNA status:
  • ND4 / ND1 → indicator of mtDNA deletion load
  • D-Loop / ND1 → indicator of mtDNA integrity and replication
  • ND1 / B2M → indicator of mtDNA copy number per cell
20m