qPCR vs dPCR: Choosing the Right PCR Technology To Suit Your Experimental Needs
Explore how you can identify which technique is best suited to your research requirements.
Complete the form below to unlock access to ALL audio articles.
Quantitative PCR (qPCR) and digital PCR (dPCR) both provide sensitive and specific detection, and precise quantification of nucleic acids. These technologies have numerous applications across genomics research, from rare cell detection in liquid biopsies to pathogen quantification in wastewater samples.
But how can you identify which technique is best suited to your research requirements? Here, we explore the advantages of each, considering specific applications and deliverable metrics to help you choose the right technology to suit your experimental needs.
An introduction to PCR workflows
qPCR is a well-established technology for rapid, sensitive gene expression analysis. By incorporating fluorescent reporter molecules, the accumulation of DNA product during a PCR reaction can be measured in real-time. Comparison to a standard curve – which plots the known concentrations of target DNA template against the corresponding fluorescence signal – enables quantification of the number of original copies of template DNA.
dPCR builds on traditional PCR techniques to provide absolute quantification of RNA transcripts by partitioning samples into thousands of nano-reactions, where PCR amplification of the template DNA takes place in every reaction site. Therefore, a single sample can generate thousands of data points, enabling statistical analysis of the results without the need for standard curves.
Table 1: The strengths of dPCR and qPCR.
|
| dPCR | qPCR |
| Target detection | Quantifies rare targets in complex backgrounds. | Broad dynamic range – measures low and high expression levels in the same reaction. |
| Detection of fold changes | Detects small fold changes: measure a difference of less than 100% expression (2-fold) such as 50%, or 10%. | Measure a greater than 100% (2-fold) expression difference. |
| Throughput capabilities | Up to 96-well throughput. | High throughput with 384-well format and automation. |
| Cost-effectiveness | Robust quantification with higher start-up costs. | Economical running costs with low reagent requirements. |
| Quantification | Provides absolute quantifications without the need for a standard curve. Can be used to create nucleic acid standards for qPCR. | Provides relative quantification against a reference curve. |
Cost
Traditional qPCR assays typically require lower volumes of reagents compared to dPCR, and multiplexing is possible with qPCR through the use of a good supermix and an optimized assay.
However, dPCR overcomes qPCR’s amplification efficiency issues, making it easier to multiplex and ultimately lowering its cost per reaction. Moreover, in situations where there is limited sample material or rare targets, dPCR can be cost-effective in the long term. Overall, the cost-effectiveness of a specific qPCR or dPCR method depends on the researcher's experimental requirements.
Absolute quantification
A key advantage of dPCR is its ability to provide absolute quantification without the need for a standard reference curve, while qPCR only offers relative quantification. Combining limiting dilution, endpoint PCR and Poisson statistics, dPCR enables an absolute measurement of nucleic acid concentration. This makes dPCR the most suitable option for many applications, including liquid biopsies, rare sequence detection and contaminant detection.
High-throughput screening
With the ability to run assays in 384-well plates, qPCR is well-suited to high-throughput applications, compared to dPCR, which is only capable of analyzing up to 96 samples at a time. This makes qPCR a great option for high-throughput screening, enabling the analysis of large volumes of genes simultaneously. It also offers more opportunities for automation, potentially in field-deployable devices.
Selecting the optimal PCR technology for your application
PCR techniques have an enormous range of applications. Apart from its conventional application in gene expression studies, qPCR can also be used for genotyping applications, and is currently used for molecular diagnostic applications as well. The selection of PCR technology depends on the specific results desired for each application.
Detecting rare and low-abundance targets
Given its high specificity, dPCR is recommended for the detection of mutations and single-nucleotide polymorphisms (SNPs), particularly when the mutation frequency is known to be below 1%. dPCR can detect rare mutations on wild-type backgrounds, haplotypes, and low-abundance targets in liquid biopsies, such as circulating tumor cells and cell-free DNA. dPCR is also well suited for the analysis of gene editing due to the low frequency of edits, both desired and off-target events.
Whole genome analysis
qPCR is the most appropriate technology for analysis of the whole genome, owing to its large dynamic range, ability to quantify different expression levels and discriminate splice variants. qPCR’s suitability for high-throughput applications and automation means that it can support large-scale screening studies in a cost-effective manner.
Monitoring ecosystem diversity
While qPCR has historically been used for the quantification and monitoring of bacterial and fungal populations in diverse ecosystems, dPCR offers increased precision, repeatability, sensitivity and stability. dPCR shows insensitivity to PCR inhibitors and increased tolerance to impurities, which are highly prevalent in environmental samples, enabling increased sensitivity and accurate calculation of fungi to bacteria ratios.
Wastewater surveillance
dPCR has proven superior in the testing of wastewater samples for infectious diseases. Wastewater-based epidemiology (WBE) has successfully been used to monitor diseases such as polio, hepatitis A, norovirus and, more recently, SARS-CoV-2, with shed viruses detected in wastewater samples before symptoms appear.
One of the main challenges of WBE is that samples contain large volumes of contaminants. However, by partitioning samples in dPCR, the detrimental effect of PCR inhibitors is reduced, enabling accurate detection and quantification of pathogens.
Infectious disease diagnostics
For infectious disease diagnostics, qPCR is commonly used due to its ability to accommodate a broad range of sample concentrations in a single well, and its high-throughput capabilities. For example, reverse transcription qPCR (RT-qPCR) effectively detected SARS-CoV-2 RNA in nasopharyngeal specimens, reducing the need for RNA extraction and thereby minimizing costs and turnaround times. This high-throughput approach expands testing capacity for pathogen detection in resource-limited countries.
However, for tests that need to analyze multiple targets in the same reaction simultaneously, the multiplexing capabilities of dPCR enable the quantification of many targets in a single experiment, without compromising assay performance. While it is possible to obtain the same results through qPCR, this multiplexed format may require additional optimization of primer and probe combinations.
Looking for content on PCR?
Visit this Topic Page to find a variety of curated content on PCR.
Conclusion
While both qPCR and dPCR facilitate sensitive detection and precise quantification of nucleic acids, each has distinct advantages for different applications. For large-scale screening, qPCR enables high-throughput analysis across a wide dynamic range, while dPCR is most appropriate for rare sequence detection against a wild-type background due to its ability to provide absolute quantification.
With the development of innovative new technologies, PCR will remain an integral part of gene expression workflows. Droplet-based digital PCR (ddPCR) reactions take place in individual droplets, offering great potential for use in primary care settings.
Multiplexing PCR (mPCR) combines multiple primer sets in one reaction to detect several DNA or RNA targets simultaneously, which is useful for the detection of infectious diseases. The continuous development of PCR to offer advanced insights into the genome will ensure the future of the technique.
To select the right PCR system for your research, try using a PCR selector tool.
About the authors:
Jennifer Placek is a global product manager at Bio-Rad, responsible for the real-time instrument and custom PCR plastics portfolios. She holds an MS in Cell and Molecular Biology from San Francisco State University and a BA in Integrative Biology from the University of Illinois. With prior experience in life science sales at Pall and VWR, Jennifer brings valuable insights into customer needs and market dynamics.
Justin Barker is a senior global product manager at Bio-Rad, responsible for the QX ONE Droplet Digital PCR System. Justin manages, grows and develops product solutions for life science markets, from research to applied/industrial and diagnostic.
