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Digital PCR

Absolute, not relative quantification

Choosing dPCR vs. qPCR
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Explore the benefits and see if it’s right for your research goals

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."

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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.

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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 ≥ 0.1% (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.

Rare mutation detection Copy number variation analysis Gene expression analysis miRNA expression analysis Microbial pathogen detection Viral load quantification Liquid biopsy GMO detection Genome edit detection NGS library quantification and validation Residual host cell quantification qPCR dPCR Applications Suitability level
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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.
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Researchers describe the benefits of dPCR over qPCR
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See examples of a few studies that used dPCR to overcome the limitations of qPCR.

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.
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Transitioning from qPCR to dPCR
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The QIAcuity Nanoplate Digital PCR System makes it accessible and more affordable than ever to bring the power of dPCR to your lab.

Featured webinars

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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FAQs about transitioning from qPCR to dPCR