Digital PCR workflow and products

The QIAcuity Digital PCR System is a microfluidic nanoplate-based digital PCR (dPCR) platform that enables all the necessary steps for dPCR - partitioning, thermal cycling, and imaging/data acquisition - on a single instrument.

QIAcuity advantages

Scalability

Low- to high-throughput instrument configurations – 1-, 4- and 8-plate. Up to 2 integrated thermocyclers.

Throughput
Samples processed in a work day can increase from 96 to 1248 samples depending on the plate (24- and 96-well) and instrument (1-, 4- and 8-plate) configuration used.
Multiplexing

Up to 5 detection channels (2-plex and 5-plex; exclusive of reference).

Time to result

Simple and rapid plate-based workflow takes users from sample to result in under 2 hours.

pcr dpcr qiacuity workflow

How does QIAcuity work?

Just like in qPCR experiments, sample preparation includes the transfer of master mix, probes and primers to a 8-, 24- or 96-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 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. This analysis can also be extended to remote computers within the same local area network (LAN).

Step-by-step tutorial

Getting started with QIAcuity Nanoplate dPCR can be easier than you expect. Designed for beginners, this detailed tutorial video discusses experimental considerations at each of the major steps in the workflow.
Refer to the QIAcuity Guide for more information on setting up experiments for your research applications.

The QIAcuity ecosystem

While using a QIAcuity Digital PCR System is a great foundation, it takes an ecosystem of supporting accessories, kits and assays for your digital PCR reaction to truly flourish.

You use different strategies to analyze gene expression, rare mutation detection and microbial quantification. Why struggle to find the right tools to turn those strategies into actionable insights? Explore the many options available in the QIAcuity ecosystem to customize your digital PCR runs to your specific application.

Streamline your clinical PCR workflows with QIAcuityDx

QIAcuityDx is tailored for IVD applications. This fully automated system enhances diagnostic precision and operational efficiency by reducing hands-on time and ensuring accurate detection and quantification of important genetic variations. Easily develop your own assay menu* by using QIAcuityDx utility mode and IVD medical device consumables, reagents and software.

Why multiplex with digital PCR?
Because you can save time and reagents and reduce errors by analyzing many targets simultaneously. It’s also not as difficult as it sounds.
Learn more
Didn’t find the dPCR assay you’re looking for?
Don’t despair. Find thousands of targets in microbial and mutation detection and gene expression analysis in our pre-designed assays on GeneGlobe. Or design your own assays.
Explore GeneGlobe
Expert Custom Assay Design
Expert Custom Assay Design
Let us do all the hard work. Get expert-designed custom single or multiplex dPCR and PCR assays for mutation detection, CNV analysis, gene expression and species detection.
Learn how
Front-end automation on the QIAgility
To overcome potential pipetting mistakes in setting up the QIAcuity Nanoplates for dPCR analysis, we developed a method for the front-end automation of nanoplate setup using the QIAgility liquid handling instrument.
Find out more

How to choose a QIAcuity Digital PCR System

Together with its nanoplates, kits and assays, each instrument delivers high performance and data quality, with flexible throughput and multiplexing. Ask the right questions to evaluate your needs.

  • What applications will you perform most frequently?
  • Which instrumentation options best enable your scale of study?
  • What are the costs for product care beyond the initial investment?

For further guidance about comprehensive product care when making your decision, download the QIAcuity Service Brochure.

Publications

Browse a growing list of articles featuring QIAcuity dPCR products.

Scientific literature
Are there guidelines on how to set up dPCR experiments?

You may refer to our QIAcuity Application Guide for detailed guidelines and step-by-step tutorials on how to set up digital PCR experiments. We would also like you to refer to the digital MIQE guidelines and their update (Huggett JF et al, 2013 and 2020) for additional considerations when setting up and reporting results from digital PCR experiments.

Does incorrect handling of a plate affect the results?
The QIAcuity reads emitted fluorescence from the bottom of the plate, which is covered with a foil. For best results, keep the foil clean and avoid damages such as scratches. Also, keep the barcode on the side of the plate clean and intact. Ensure that you wear gloves when working with a plate and do not apply force to it. For safe handling of the plate, please place the plate into a nanoplate tray.
How to analyze dPCR data?
Your dPCR system software will likely provide you with first-level and second-level analyses. First-level analysis could include a concentration diagram, a view of the partitions in wells of interest or a heatmap to compare the target channel to a reference channel. You can use histograms and scatterplots to change threshold settings. In second-level analysis, you can analyze PCR data according to application aims (mutation detection, gene expression analysis, etc.).
Are there any special considerations for sample input amount in dPCR experiments?

High amounts of gDNA will result in increased background fluorescence in EvaGreen. Usually, 100 ng will be more than sufficient for most analyses. 

Given that the haploid genome size of the organism studied is known, the correlation between mass input of gDNA and the resulting copy number (for a single-copy gene) can be easily calculated using the following formula: 

 

Size of the genome (bp) x average weight of a single base pair (1.096 x 10–21 g/bp)

 

In dPCR, the average number of copies/partitions must not exceed 5, ideally in the range of 0.5–3. Suppose the copy number input cannot be determined before starting the experiment. In that case, it is recommended to perform an initial titration experiment, using the unknown template in 2–4-fold dilutions to determine an optimal range for subsequent analyses. You may refer to our QIAcuity Application Guide for more detailed sample preparation guidance.

How to design primers for digital PCR?
Designing primers for digital PCR is similar to designing primers for qPCR. You should pay attention to target matching, base composition, amplicon length, melting temperature, secondary structures, self- and inter-complementarity (self-annealing) and cross reactivity. One difference is that primers for dPCR are often used at higher concentrations than qPCR to better separate specific signals from background noise.
When does one need to perform restriction digestion for a dPCR experiment?
For the vast majority of QIAcuity dPCR applications, template DNA is uniformly distributed throughout the QIAcuity Nanoplate reaction chambers. In QIAcuity reactions using PCR products or highly fragmented DNA (FFPE DNA, circulating cell-free DNA), complementary DNA (cDNA), gBlocks, etc., as a template, a uniform distribution of PCR signal is observed. However, DNA molecules >20 kb or plasmids are unevenly partitioned, which leads to over quantification of template concentration. By adding restriction enzymes directly to the QIAcuity reaction mixes, large templates can be fragmented to smaller sizes, which results in even template distribution and accurate quantification. This is particularly important for copy number variation (CNV) analyses, where multiple copies of a gene might be linked in tandem. When adding restriction enzymes to reaction mixes, users must be sure that the enzymes do not cut within the amplicon sequence. It is recommended to include restriction enzyme in the master mix and incubate for 10 min at room temperature after template addition.
Which dyes can I use?
The QIAcuity dPCR instrument can detect dyes such as FAM, EvaGreen, VIC, HEX, TAMRA, ROX and Cy5. 
How to prepare DNA before dPCR?
All DNA samples used in reaction mixes should show similar quality and quantity, which can easily be assessed using UV spectrophotometry. DNA samples with an average length of ≥20 kb (e.g., genomic DNA purified via spin column with silica-membrane) should be fragmented by restriction digestion before partitioning. Enzymatic fragmentation of larger DNA ensures an even distribution of template throughout the QIAcuity Nanoplate, which in turn leads to an accurate and precise quantification.

*FDA ‘Medical Devices; Laboratory Developed Tests’ final rule, May 6, 2024 and European Union regulation requirements on ‘In-House Assays’ (Regulation (EU) 2017/746 -IVDR- Art. 5(5))

The QIAcuity dPCR System is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease. Therefore, the performance characteristics of the product for clinical use (i.e., diagnostic, prognostic, therapeutic or blood banking) is unknown.

The QIAcuityDx dPCR System is intended for in vitro diagnostic use, using automated multiplex quantification dPCR technology, for the purpose of providing diagnostic information concerning pathological states.

QIAcuity and QIAcuityDx dPCR instruments are sold under license from Bio-Rad Laboratories, Inc. and exclude rights for use with pediatric applications.

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