QIAcuity, digital PCD, dPCR, Labworker handling the instrument, loading tips, Chaimae Safi, Amit Singh, mask, wearing masks, labcoat
September 15, 2023 | PCR Solutions

Five tips on how to design your first (or 15th) dPCR multiplex assay


Do you know how to peel an avocado? Most likely, at some point in your life, you held an avocado for the first time and managed to free it of its skin. Then you probably stuck to your unglamorous cutting protocol for years to come. Until your neighbor came over for dinner, watched you butcher the avocado and showed you a better way to do it. 

We’re here to be your friendly neighbor, but we're not here to help you slice though avocados. Rather, we'll help you overcome challenges in multiplex dPCR. Whether you’re just starting your first design or think you’ve already mastered multiplexing, these five tips will give your assay development a cutting-edge.

Tip 1: Decide if you need a dPCR multiplex assay

Singleplex dPCR might be enough to answer your research questions. You’d save yourself the time of designing and developing a dPCR multiplex assay. But in many cases, it’s worth investing some brain and experimental power into multiplex dPCR. Even though the design of multiplex dPCR might appear challenging, a dPCR multiplexing approach will save you time, reagents and costs in the long run.

Why else would you use a dPCR multiplex assay? If you’re working with low-copy and rare targets for example, analyzing multiple targets in a single reaction increases your chances of detecting the target. If you’re working with precious sample, you can reduce the volume of sample you need by using a multiplex dPCR approach. 

A dPCR multiplex assay also allows you to use a direct internal control of an individual reaction for more reliable results. Digital PCR multiplexing can also help improve the accuracy of your data by reducing errors, such as pipetting inaccuracies. 

Tip 2: Weigh in the different multiplex PCR approaches

You have various options for how to design a multiplex PCR assay. You can try using traditional multiplex qPCR, but qPCR multiplexing might be more susceptible to the interference of multiple PCRs in one reaction. This could be especially problematic when analyzing complex samples containing PCR inhibitors or detecting rare targets (1).

Detection of rare targets is one of the specialties of nanoplate digital PCR, including multiplex dPCR. Because you analyze individual PCR reactions rather than bulk droplets, you can find rare targets (<10-20 copies/reaction) even in dPCR multiplex assays and in a variety of sample matrices (1).

Tip 3. Adapt multiplex dPCR to your application

Depending on your sample and research goal, your dPCR multiplex assay is designed to serve a different purpose. Tying into tip 1, multiplex dPCR can offer application-specific benefits.

For example, if you’re performing copy number variation analysis, you can design a multiplex assay to determine pair target and reference gene ratios. You can use this approach to detect rare events, gene mutations, fusions or duplications or for genotyping purposes (2).

If you’d like to detect bacterial, fungi, viruses or resistance genes, multiplex digital PCR can deliver accuracy and precision (3–6). One of the benefits of using a multiplex dPCR assay over qPCR is higher resistance to PCR inhibition, which is common in environmental samples. Digital PCR multiplexing is also better than qPCR at quantifying multiple targets with large-fold differences in concentration (7). For more, check out a great poster on multiplexing up to five microbial targets

Researchers in cell and gene therapy can use multiplex dPCR assays in manufacturing and quality control, for example by quantifying and qualifying adeno-associated virus (AAV). You can readily evaluate vector genome integrity by using multiplex assays. As a result, you gain a higher yield of full-length viral genomes in capsids and a well-characterized, safe product (8). With dPCR multiplexing, you can also obtain more information from a single sample, reducing the need for re-sampling, as well as the costs per reaction.

Find out more about how to use multiplex dPCR in single-cell analysis, multiplex dPCR for CAR-T products and multiplex dPCR in cosmetics in our dedicated webinars. 

Tip 4. Think about the factors that can affect your multiplex dPCR assay

When you start designing your dPCR multiplex assay, consider several factors. Like any PCR reaction, you want to avoid primer dimers and any potential interaction between your primers and probes. To avoid cross-hybridization, try designing probe-to-target variations with two or more nucleotide deletions or insertions to avoid probe mismatches. 

Validate single reactions before moving on to multiplex dPCR. Validation might appear time-consuming, but it can save you plenty of time, money and headaches in the long run (9).  

Make sure you purify your nucleic acid template carefully. If your template's type, confirmation or integrity is suboptimal, you could see rain in your final data. This unwanted phenomenon could also result from non-specific assays or from the presence of inhibitors in the reaction (9).

Select your dyes carefully. A list of available dyes on the QIAcuity Digital PCR System is available below:

 Channel  Excitation (nm)   Emission (nm)   Example fluorophores 
 Green  463 – 503   518 – 548   FAM 
 Yellow  514 – 535  550 – 564   HEX, VIC 
 Orange  543 – 565   580 – 606   TAMRA, ATTO 550
 Red  570 – 596   611 – 653  ROX, Texas Red
 Crimson  590 – 640   654 – 692   Cy5 


Sometimes, you can improve your results by making adjustments with the analysis software. For example, you can alter signal processing parameters to reduce optical bleedthrough. Or you could set the threshold position differently to avoid the influence of rain on your quantification.

Tip 5. Research instruments and assays that can make your life easier

You might start with just duplex assays, and before you know it, you need to analyze more targets at once. Be prepared for all possible scenarios by knowing what dPCR multiplexing capabilities different digital PCR systems can offer. For example, with the QIAcuity Digital PCR System, you can multiplex up to five targets and one control at one go. It’s about as flexible as you can get.

You might also be a fan of DIY projects, but sometimes it’s easier to go to IKEA than build that cupboard from wood you chopped in the forest. Application-specific assays can ease your entry into the dPCR multiplexing world or make your stay there more comfortable. 

For example, if you’re working on copy number variation (CNV) analysis, there are multiplex CNV assays for that. If you’re interested in microbial targets, there’s a microbial detection assay for analysis of up to five targets at once. There are also cell and gene therapy multiplex assays. And all types of kits, master mixes and other assays that could elevate your multiplex digital PCR performance.

We've got some great news if you’re looking for more details about digital PCR multiplexing. You could learn more about it from 18 advanced users of digital PCR at our Digital PCR Festival. Or you could read more about multiplex dPCR for different applications. Either way, the best way to learn is to try, so we wish you multi-success with your next dPCR multiplex assay design.

Let 18 experts teach you dPCR multiplexing
Learn from 18 advanced users as they showcase their digital PCR research for salmonella testing, HIV, cancer, cell and gene therapy and more.


  1. Center for Breakthrough Medicines. https://breakthroughmedicines.com/all/tech-note-high-throughput-aav-viral-titering-using-nanoplate-based-digital-pcr/ (Accessed September 6, 2023)
  2. Appay R et al. Multiplexed droplet digital PCR assays for the simultaneous screening of major genetic alterations in tumors of the central nervous system. Frontiers in Oncology. 2020; 10:579762.
  3. Dong L et al. A rapid multiplex assay of human malaria parasites by digital PCR. Clinica Chimica Acta. 2023; 539:70–78.
  4. Shi K et al. A multiplex crystal digital PCR for detection of African Swine Fever Virus, Classical Swine Fever Virus, and Porcine Reproductive and Respiratory Syndrome Virus. Frontiers in Veterinary Science. 2022; 9:926881.
  5. Wu, J et al. Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool. Crit Care. 2022; 26: 243.
  6. Zhu X et al. Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections. Virology Journal. 2022; 19:70.
  7. Te SH, Chen EY, Gin KYH. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria Cylindrospermopsis and Microcystis. Applied and Environmental Microbiology. 2015; 81(15):5203–5211.
  8. Dobnik D and Hayes DB. Commentary: Multiplex dPCR and SV-AUC are Promising Assays to Robustly Monitor the Critical Quality Attribute of AAV Drug Product Integrity. J Pharm Sci. 2022; 111(8):2143 – 2148. 
  9. The dMIQE Group and Hugget JF. The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020. 2020; Clin Chem 66(8):1012-1029.