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Digital PCR

Absolute, not relative quantification

When comparing dPCR vs. qPCR technologies, the key difference is precision power. While both offer highly sensitive and reliable nucleic acid detection and quantification, the key difference between the two technologies can be best described using an analog versus digital radio analogy, says Dr. Jim Huggett, Principal Scientist, National Measurement Laboratory. "With an analog radio, the dial must first be fine-tuned to get the desired station with the least interference. Still, the quality depends on reception and the signal is subject to interference from static. This is qPCR. It is reliable but requires optimization to get a good result, and even then, you must contend with background noise. With digital radio, you simply call up the station and it is either there, with a clear signal, or not. The latter is like dPCR, which provides precise, binary results. It literally counts the presence or absence of DNA molecules. The clarity of results combined with a low error rate makes for an incredibly high level of precision. Digital PCR is well-suited to measure smaller quantitative differences."

Compare and contrast: qPCR vs. dPCR

Researchers value qPCR for its speed, sensitivity, specificity, and ease of use. The technique is most useful when performing gene expression analysis, pathogen detection and microbiome analysis, as well as microarray data validation. However, qPCR is seen to falter in applications requiring superior accuracy and sensitivity, such as copy number variation analysis, detection of mutations and SNPs, as well as allelic discrimination. In such applications, dPCR outperforms qPCR by not only measuring the absolute copy number but also overcoming the limits of detection, i.e., detecting small fold-change differences expressed as 10% precision and mutation rates <1%.

Digital PCR also demonstrates robust quantification, i.e., high tolerance to PCR inhibitors and less affected by changes in PCR efficiency owing to sample partitioning and end-point cycling.

Real-time PCR/ qPCR Digital PCR
Quantitative, relative or absolute but standard
curves or reference samples needed
Quantitative, absolute and no standards or
references needed

Bulk PCR

  • flexible reaction volumes
  • impacted by changes in PCR efficiency
    as data is collected at the exponential phase
  • prone to inhibitors

Sample partitioning

  • higher inhibitor
    tolerance / increased robustness
  • unaffected by changes
    in amplification efficiency
  • higher statistical power subject
    to the Poisson statistics
Measures PCR amplification at each cycle Measures at the end of the PCR cycles
Detects mutation rate at >1% Detects mutation rate at ≥ 0.001% (high
signal-to-noise ratio)
Well-established protocols Higher precision for higher reproducibility
across laboratories

dPCR reactions remain robust even in the presence of inhibitors such as humic acid and heparin. qPCR (on Rotor-Gene Q) and dPCR (on QIAcuity) reactions were carried out in the presence of indicated amounts of the inhibitors using the respective QIAcuity PCR Master Mix (EvaGreen) and using identical reaction volumes. Quantification shows Cq (qPCR) or copies/µl (dPCR) as relative performance differences among samples with the non-inhibited sample set to 100%.
dPCR reactions remain robust even in the presence of inhibitors such as humic acid and heparin. qPCR (on Rotor-Gene Q) and dPCR (on QIAcuity) reactions were carried out in the presence of indicated amounts of the inhibitors using the respective QIAcuity PCR Master Mix (EvaGreen) and using identical reaction volumes. Quantification shows Cq (qPCR) or copies/µl (dPCR) as relative performance differences among samples with the non-inhibited sample set to 100%.
1 Cq in qPCR which represents 50% precision is equivalent to 10% precision in dPCR and corresponds to a 2x concentration increase.

When to use dPCR vs. qPCR?

When it comes to molecular biology and genomics research involving nucleic acid quantification, scientists often find themselves standing at the crossroads. Which quantification technique to choose to achieve research goals efficiently – the more precise and robust digital PCR (dPCR) or the more standardized and familiar quantitative real-time PCR (qPCR). Both technologies have similarities, but also their benefits and limitations that make the choice application-dependent.

The application grid indicates the suitability level of each technology for some of the common applications.

See what dPCR can do for you
Digital PCR and in particular the QIAGEN nanoplate-based technology is fundamentally changing the questions you can answer today, enabling a broad range of applications.
Beginner’s guide to dPCR
These resources are designed to offer valuable guidance to anyone who is considering switching to dPCR and planning their first experiment.
  • Transforming the PCR experience
    Imagine keeping the familiarity and ease of qPCR but gaining the higher sensitivity and precision of digital PCR, without prolonging the time to scientific insight. The QIAcuity, QIAGEN's fully integrated nanoplate-based digital PCR system, has been designed with your research needs and the limitations of the currently available methods in mind.
  • How does QIAGEN Nanoplate dPCR work?
    Just like in qPCR experiments, sample preparation includes the transfer of master mix, probes and primers to a 96- or 24-well nanoplate, followed by the addition of samples. The system integrates partitioning, thermocycling and imaging into a single fully automated instrument that takes users from the sample to result in under 2 hours. One can perform remote analysis on the Software Suite, providing the concentration in copies per microliter of your target sequence as well as for quality control such as positive samples or NTC.
    How does QIAGEN Nanoplate dPCR work?
  • Unveiling the magic inside
    Reimagining familiarity, ease of use and finer resolution, we have developed these nanoplates with superior partitioning, meaning unparalleled precision and sensitivity gain for your assays.
  • No droplets. No crystals. No chips.

    The key advantages of performing digital PCR in nanoplates include but are not limited to:

    - User-friendly, familiar plates are easy to pipet just like for qPCR
    - Fixed partitions prevent variation in size and coalescence
    - Sealed nanoplates prevent well to well contamination
    - Faster readout possible due to simultaneous reading of all partitions of a sample
    - Integrated quality control, meaning one can look at the plate image for single fluorescent signals from the individual positive partition counts
    - Plates are amenable to front-end automation

  • Simplifying going digital

    It's time to simplify the transition from qPCR and prepare your critical research applications to benefit from digital PCR in more ways than one. Yes, in just 3 simple steps you can have the accurate result you want in under 2 hours: pipette and load, let the experiment run, analyze results. Moreover, cost, scalability and throughput are no longer a bottleneck when considering that switch.

  • Transforming the PCR experience
  • How does QIAGEN Nanoplate dPCR work?
    How does QIAGEN Nanoplate dPCR work?
  • Unveiling the magic inside
  • No droplets. No crystals. No chips.
  • Simplifying going digital
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Digital PCR tips and tricks
Find a compilation of the top 20 questions, along with insightful answers from our guest webinar.
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