Digital PCR

dPCR vs qPCR

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 instruments instruments demonstrate 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%.

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.

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.

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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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The QIAcuity nanoplate dPCR workflow: Just like qPCR…But better

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.

FAQs about transitioning from qPCR to dPCR

Do others use nanoplate dPCR instead of qPCR? At least 300 publications in 3 years do…

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