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Digital PCR

dPCR vs qPCR

Choosing dPCR vs qPCR – 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.

Download application note
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

qpcr vs dpcr heparin
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%.
digital pcr vs qpcr humic acid
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%.
qpcr vs dpcr precision
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

Droplet digital PCR (ddPCR), one of the earliest forms of digital PCR, could already offer advantages over qPCR for most of the applications above. In ddPCR vs qPCR, qPCR is suitable for applications that require a broad dynamic range, whereas ddPCR is for those applications that require higher precision or analysis of fractional abundance.

The evolution from ddPCR to nanoplate dPCR has widened the scope of this technology to include more applications. The nanoplate dPCR workflow is substantially faster thanks to the simultaneous reading of all sample partitions, front-end automation, and an easy qPCR-like plate set-up. This added speed makes it suitable for screening and high-throughput applications without compromising precision, accuracy and sensitivity.

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.
Explore benefits

Researchers describe the benefits of dPCR over qPCR
–
See examples of a few studies that used dPCR to overcome the limitations of qPCR.

Guiding researchers with cutting edge dPCR technology
Guiding researchers with cutting edge dPCR technology
Mogens Kruhøffer, founder and CEO, BioXpedia, sees the potential for technological advances in accelerating the research from idea to bedside for the benefit of patients. Their most recent addition to the lab, the QIAcuity, will allow their customers to provide better results even faster.
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Cracking the malaria epidemiology code with dPCR​
Cracking the malaria epidemiology code with dPCR​
Switching from qPCR to dPCR enabled Dr. Wang Nguitragool to identify a few unknown samples which could not be identified by their standard assay.
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Identifying the genetics behind obesity using dPCR​
Identifying the genetics behind obesity using dPCR​
Dr. Johanna Andersson-Assarsson used dPCR to determine exact copy numbers of a highly variable obesity gene with a large number of copies as the resolution was just not good enough with qPCR.
Find out more
A new climate for vector-borne diseases
A new climate for vector-borne diseases
Climate change, travel and migration are making it easier for disease-carrying insects to spread into new areas. Even worse, many vector-borne diseases are very difficult to detect. Learn how Dr. Melissa Nolan from University of South Carolina is exploring digital PCR to help detect infections early so immediate action to save lives can be taken.
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Meet the 2021 APHL dPCR grant winner

Dr. Abelardo Moncayo, Director of the Vector-Borne Diseases Program, Tennessee Department of Health, is the 2021 recipient of QIAGEN's Digital PCR Technology Access Grant in partnership with APHL. Hear how he intends to use digital PCR in guiding research and surveillance strategies against mosquitoes and ticks.
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
–
The QIAcuity Nanoplate Digital PCR System makes it accessible and more affordable than ever to bring the power of dPCR to your lab.

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QIAGEN’s new dPCR portfolio – overcoming the current throughput, speed and usability bottlenecks in digital PCR
QIAGEN’s new dPCR portfolio – overcoming the current throughput, speed and usability bottlenecks in digital PCR
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FAQs about transitioning from qPCR to dPCR

How is the assay used in dPCR different from the assay used in qPCR?
All the rules for proper real-time PCR assay design also apply to dPCR. An assay working well in real-time PCR will most likely do so as well in dPCR. One can easily transfer SYBR assays to EvaGreen dPCR. However, care should be taken that the recommended cycling conditions and primer/probe concentrations for dPCR, as outlined in the respective QIAcuity dPCR Kits, are selected. For fast optimization of suboptimal performing assays, for example, by running a temperature gradient during the annealing steps, the QIAcuity master mixes can also be run on any real-time PCR instrument. You may refer to our QIAcuity Application Guide for more detailed assay design guidance.
What is the difference between qPCR and dPCR?

The difference between qPCR and dPCR is how the methods quantify nucleic acids. qPCR measures amplification as it occurs and uses standard curves to determine the amount of nucleic acid in the sample. dPCR divides the sample into thousands of partitions, collects data at the end of each reaction and uses statistics to obtain the absolute number of molecules in each reaction.

What is the difference between digital PCR and RT qPCR?
The difference between digital PCR and RT qPCR is the quantification method. In any RT-PCR, an RNA sample is reverse transcribed into cDNA, which is then amplified in a PCR reaction. In RT-qPCR, the cDNA is quantified in real time using standard curves. In RT-dPCR, cDNA is measured at the end of the reaction and Poisson statistics are used to achieve absolute quantification. You can use both methods to analyze RNA and gene expression.
Is a standard curve needed in dPCR?
In dPCR, we measure the absolute concentration of targets at the endpoint reaction. Concentrations of unknowns can be determined based on dPCR results observed (number of negatives, number of positives and partition volume analyzed).
What are the advantages of digital PCR over qPCR?
Digital PCR is often preferred over qPCR because dPCR does not require calibration curves and is unaffected by differences between the calibrant and sample. Digital PCR has high PCR efficiency, is very precise and suitable for detecting rare targets in a high background of non-target DNA. dPCR is also less sensitive to PCR inhibitors than qPCR.
What is a qPCR used for?

qPCR is used to detect rare mutations(ctDNA), rare sequences (viral and bacterial load, miRNA), copy number variations, to quantify gene expression, for SNP genotyping, to verify microarrays, validate assays, detect pathogens and quantify NGS libraries, among others.

What is qPCR and how does it work?
Quantitative PCR (qPCR) is a method for quantifying nucleic acids. qPCR works by measuring DNA amplification in real time through monitoring fluorescence. At a certain point, the fluorescence intensity increases proportionally to the initial number of template nucleic acids in the sample. The quantity of target DNA is calculated using a calibration curve made from serial dilutions of a standard with known concentrations.
What is the disadvantage of qPCR?
The disadvantage of qPCR is that the method relies on standard curves. The need for these curves costs time, resources and negatively impacts PCR efficiency. The qPCR method is less precise, less reproducible and more sensitive to PCR inhibitors and contaminants than dPCR.
Can I use the dPCR master mix in a qPCR cycler for optimization purposes?
dPCR master mix can be used in qPCR to optimize sample concentration and/or primer/probe concentration before assay transfer from qPCR to dPCR.