Are qPCR and digital PCR competing technologies or complementary tools? In this Science Snapshot, we revisit conversations with researchers across multiple disciplines to explore how each PCR method brings unique strengths, from high-throughput screening to ultra-sensitive absolute quantification.
Choosing between qPCR and digital PCR isn't about finding a winner, it's about asking the right scientific question.
In this Science Snapshot, Jordan Ruggieri and Lisa Crawford revisit insights from researchers including Corbin Schuster, Nikhil Ram Mohan, Brandi Kiel Reese, Pavla Brachova, Nehemiah Seth Alvarez, Raquel Muñoz, Rounak Feigelman, and Brian Bahder to explore how qPCR and dPCR work together across a wide range of applications. From environmental DNA and pathogen surveillance to COVID viral RNAemia, reproductive biology, CAR-T cell monitoring, and assay development, each guest demonstrates why qPCR often provides the speed, flexibility, and throughput needed for discovery, while digital PCR delivers the analytical sensitivity, precision, and absolute quantification required for challenging targets. Together, these examples illustrate that the strongest workflows often combine both technologies, allowing researchers to move seamlessly from broad screening to highly confident quantitative measurements.
Lisa Crawford 00:00
All right, are we ready to do this?
Jordan Ruggieri 00:02
I'm going to change my second line to say, “and I'm Jordan Ruggieri. Today we're exploring why we can't get through even one line of a recorded podcast.”
Lisa Crawford 00:21
Welcome to another Science Snapshot episode of the Applied Biosystems Absolute Gene-ius podcast by Thermo Fisher Scientific. I'm Lisa Crawford.
Jordan Ruggieri 00:30
And I'm Jordan Ruggeri. Today we're exploring a theme that surfaced in so many conversations in this series, which is how qPCR and dPCR aren't rivals, but instead teammates.
Lisa Crawford 00:41
We've spoken to scientists across fields like virology, ecology, reproductive biology, and cancer immunotherapy research, and have noticed a common theme: qPCR gets you breadth, dPCR gets you depth.
Jordan Ruggieri 00:54
So today, we're weaving together some of our favorite moments from past episodes, highlights that illustrate how these technologies complement each other in powerful and important ways.
Lisa Crawford 01:04
Our first clip with Dr. Corbin Schuster takes us into the world of zebrafish as a model organism and their work to monitor zebrafish health. His team starts with building qPCR assays as a fast, flexible, and easy to optimize monitoring tool.
Jordan Ruggieri 01:18
As Corbin explains, even qPCR has its limits, particularly when the goal is to identify barely detectable pathogens. This is when his team finds that dPCR really shines in detecting low-level infections that may otherwise be missed by qPCR.
Corbin Schuster, PhD 01:35
The nice thing with qPCR is it's essentially we will build our qPCR assays, and that's what we will use to, you know, do detection of these pathogens for tissues and stuff in different sorts of tissues. Pseudoloma neurophilia specifically targets the central nervous system, so looking at the hindbrain and like spinal cord tissue, or in the spinal column, and so it's really nice. That's sort of where we start, and then it's a very easy transition to take our qPCR assay and you know take it to the digital PCR platform. It's essentially the same primers, a little different master mix, but you know we can take our primers and our probes and directly translate that over to digital PCR. Where I think the digital PCR has really complemented our qPCR efforts has been in the fact that we can detect the pathogen at much lower concentrations than what we would have with the qPCR, especially what we've seen with environmental sampling. We've used our qPCR assay for environmental samples, whether that be like tank detritus or biofilms or straight tank water. It was very inconsistent. We could detect it if the pathogen had a really high concentration in an environment, but oftentimes with Pseudoloma neurophilia, there's both extracellular, so you know outside of the cell, but it's an intracellular parasite. So there's also intracellular phases, especially as that moves essentially through every bodily organ system until it finally reaches its quote final destination to the spinal cord and or you know the spinal column. So that movement, we did transmission studies, sort of looking at what does that look like actually for the detecting the parasite in the environment. And what we found was really during those initial intracellular stages, it's not really as it's moving through those organ systems within the host, there's some you know there's some gaps in detection, and so that gap in detection, you know, it's very it's varied. It varies by how big your population is, how big of a, you know, inoculum or a dose that the actual fish are exposed to. So being able to detect it at very low concentrations was very important, and the only way we were able to truly achieve that was with the digital PCR.
Lisa Crawford 03:48
Corbin's workflow is one we have heard time and time again from our guests. qPCR excels for development or for targets in abundance, and dPCR excels for its analytical sensitivity.
Jordan Ruggieri 03:59
Another example about the complementary use of these technologies comes from Dr. Nikhil Ram Mohan. Nikhil's work focused on viral RNAemia in COVID, which is when the virus RNA presence in a subject's blood indicates the infection has spread beyond the respiratory tract and becomes more serious.
Lisa Crawford 04:17
He ran qPCR and dPCR side by side and identified remarkable differences in results. Using qPCR he detected viral RNA in only about 1% of samples. When switching to dPCR, viral RNA was detected in 24% of samples. Definitely not a small difference.
Nikhil Ram Mohan, PhD 04:35
When we were studying the viral RNAemia with qPCR, we were finding that, of the subjects that we had tested, there was a very low positivity rate, but we didn't know if that was because of the lack of sensitivity in, sensitivity in the qPCR mechanism itself, or it was actually the case, right. So we started testing out the digital PCR, and we saw that if I remember the numbers correctly, with qPCR, our positivity rate was about 1.4 percent, but with digital PCR, we saw that they jumped up to about 24 percent, and we are getting absolute quantities, right. So there's no need for a standard curve or anything to get the viral load. So it just made more sense for us to go that route for that particular study.
Jordan Ruggieri 05:19
In terms of that huge jump from 1% to 24% How did that how did that you know impact the maybe the precision or the observations for the for your study?
Nikhil Ram Mohan, PhD 05:31
The challenge was that a lot of the samples that we had had really low viral loads to begin with. If we had relied on the 1% from qPCR for us to go forward in that study, we then needed a larger end for us to be able to do further analyses, right. But with a digital PCR, that helped us out because the rate-limiting step there was that we were only able to sample so many subjects coming in as well. The higher sensitivity with the digital PCR platform really helped us in being able to ascertain the patterns that were actually present, which we would have missed out relying on just the qPCR.
Jordan Ruggieri 06:14
I absolutely love that Nikhil's study relied on both qPCR and dPCR. Both results were key to the study, while also demonstrating how the analytical sensitivity of digital PCR can be critical for certain applications. Same samples, same primers, same day, significantly different results, but both valid and useful.
Lisa Crawford 06:36
Moving on from the controlled environment of hospital-based research, our next clip reiterates this point. Dr. Brandi Kiel Reese studies microbes in some of the most extreme, lowest biomass environments on Earth, from the Mariana Trench to Antarctic sediments.
Jordan Ruggieri 06:50
Brandi explained how qPCR and dPCR complement each other when working with samples from the bottom of the Mariana Trench, attempting to detect RNA and DNA from dormant organisms.
Brandi Kiel Reese, PhD 07:01
If we want to look at specific genes or specific transcripts of interest that might be associated with a particular metabolism or a secondary metabolite, something like that, then we'll go into it further with quantitative PCR or digital PCR. And then also we take you know some of those and we try to grow them up in the lab. We take, you know, sequencing both the DNA through metagenomics, the RNA through metatranscriptomics. The quantitative PCR allows us to quantify, you know, how active a gene might be, maybe through looking at its gene expression, doing qPCR on, you know, on the on the transcripts themselves. And we often run into the problem of that we have such low biomass environment that we have to look at it a little bit more specifically. The qPCR is a great tool if you have a thousand to a million copies already, then it's great. It's in its sweet spot. But if you only if you're starting out with only thousand cells per gram of sediment, then you're only going to have you know maybe tens of copies or just single copies of that particular gene or transcript. So that's where digital PCR is much more useful, and we're able to use that to get very low detection, and that technology has enabled us to go further within the analyses.
Lisa Crawford 08:30
Another tag team win for qPCR and dPCR. qPCR being the workhorse through broad dynamic range and throughput, and dPCR finishing the job with its ability to detect rare transcripts in samples where biomass is almost non-existent.
Jordan Ruggieri 08:44
And then in season two, we talked to doctors Pavla Brachova and Nehemiah Seth Alvarez, whose research focuses on understanding the role of RNA degradation in oocytes.
Lisa Crawford 08:54
They used qPCR to help design and validate their assays. When it came time to determine the exact number of RNA molecules in a single cell, dPCR provided the precise analytical sensitivity they needed.
Pavla Brachova, PhD 09:06
In the past, I've done a lot of qPCR in graduate school and in postdoc, and it's always the results and the interpretation of those results, like what does it actually mean, depends on which gene you've chosen, which gene other literature has, like other papers have used to use as your reference, yeah, to make it relative to that gene. But it's really unknown how that reference gene is changing over time, how your experimental conditions are impacted, and so it makes it more murky, and it complicates the interpretation, and then just the overall entire experiment. So that's another reason why it makes it so much more powerful, just to quantify the absolute amount.
Nehemiah Seth Alvarez, PhD 09:59
By the nature of what we look at, we use a lot of single cell sequencing technology approaches, and so this is either through actually the sequencing of the direct you know of the oocyte itself, or doing you know some sort of you know using a single cell sequencing platform, so we can look at potentially what those like within the whole tissue, what those oocytes are looking like transcriptionally within the tissue, you know, not after we have like manually taken them out and you know and manipulated them in some way. Within the ovary, look at you know single cells within that tissue, and we look at the oocytes, and then we also look at these oocytes in isolation using just a standard sequencing platform, and this just gives us a nice large snapshot of what that transcriptional environment looks like, and gives us the ability to look at targets that then we can interrogate in much more, you know, much more precise detail using the you know digital PCR.
Lisa Crawford 10:58
qPCR helped these researchers create a foundational base for their experiments, providing great jumping-off points for more in-depth questioning. dPCR gave them the absolute quantification and details they were looking for.
Jordan Ruggieri 11:09
Besides the analytical sensitivity advantages, dPCR also provides improved resistance to PCR competition and inhibitors. This advantage within the complementary nature of these PCR methods is explained well by immunologist Dr. Raquel Munoz, who studies CAR-T cell expansion dynamics in leukemia subjects. She explains how qPCR struggled to quantify CAR-T cells at low abundance, especially when reference genes outcompeted the target.
Lisa Crawford 11:37
With digital PCR, Raquel could finally measure CAR-T expansion curves with confidence. No need for a standard curve. No competition, just absolute counts.
Raquel Munoz, PhD 11:48
We tried to measure the expansion of CAR-T cell first with a real-time PCR, but we had a lot of problems because, as you know, with the real-time PCR, we need to amplify a reference target, and in the same reaction, we have all the reagents and the primers to amplify our target, but also the target reference, the reference target. Sorry. So in cases of low frequency targets, as our case, there is a high probability of competition between the reagents, and maybe we could amplify only the reference target because there is a high proportion if we compare with our target, so this problem doesn't exist with digital PCR, because thanks to partitioning we have micro amplifications. So we do an absolute quantification. We don't need a reference, so we obtain an absolute quantification, and in our research, this is a great advantage. So we decide to use digital PCR because with real-time PCR, in in some cases we didn't, we wasn't able to amplify our target because there was very low proportions. With digital PCR, we are sure that if we don't see amplification, we are in the third group that I am, as I said before. But maybe with real-time PCR, we are not sure that if we are in the third group and there is no amplification, there is no expansion, or maybe there are competition between the reference and the target. So using digital PCR, we are sure about this.
Jordan Ruggieri 14:00
Here's another clip that touches on these types of considerations. Specifically, we spoke with Thermo Fisher Scientific assay designer Dr. Rownak Feiglman, who touches on similarities and differences in assay design for qPCR and dPCR, and how reaction efficiency can come into play. Let's listen.
Rounak Feigelman, PhD 14:19
Even if you design an assay for qPCR you can try running it on digital PCR and see whether it performs well. A lot of times it will perform well, but there are some considerations for digital PCR. Digital PCR, the readout is at the endpoint, right. And in qPCR, you have like a real-time readout, and so sometimes when an assay does not perform so well in qPCR, you might see that it still performs well in digital PCR because the readout is at the endpoint. Design considerations differ slightly. But overall, I do see that like the transference of an assay from qPCR to digital PCR is quite high.
Jordan Ruggieri 15:07
So, for digital PCR, does reaction efficiency play as large of a role in like say it does in qPCR, in real time PCR?
Rounak Feigelman, PhD 15:18
It does, right. Because you have very few templates in every chamber, so with that respect, like you know, reaction efficiency would be like a key factor in making sure that like, you know, we do get the signal that we are expecting towards the end. And since we do take pride on the fact that like you know we can detect like low copy number targets that also makes it important that the reaction efficiency is good.
Lisa Crawford 15:49
Great to hear the high transference rate of assays between methods, which is really helpful in supporting the complementary nature we've been hearing about in earlier clips. And the reaction efficiency point is a great one that probably doesn't get considered as often as it should.
Jordan Ruggieri 16:03
I agree, Lisa. Let's close with one last clip on the complementary nature of qPCR and dPCR. I love this clip from when we spoke with entomologist Dr. Brian Bader about how he uses both technologies. It's a perfect way to summarize and close on how these technologies are not mutually exclusive.
Lisa Crawford 16:22
Brian tracks invasive snakes and fish using environmental DNA, where targets can be rare and degraded. qPCR and dPCR are both tools in the same toolbox to assure that he gets reliable results.
Brian Bahder, PhD 16:33
Early on, I see that using qPCR with digital, depending on what you need, like if you're dealing with research questions or you need the high sensitivity, yeah, the digital PCR it's worth taking the time to do that. But if you're running like a diagnostic service where you need to get results like quickly, you can somewhat sacrifice the sensitivity because you need and qPCR is, for a lot of stuff the sensitivity is just fine. There's no problem with it, and it's faster just because of the setup. With this new system, as long as you have the, I guess the funding and support and staff, the workflow of setting up the digital PCR plate with how it's currently designed is the difference with q, it still might be a little slower than qPCR, but it's so close that in my mind, with this system, you could do away with qPCR pretty much for any research purposes. The only scenario, you know, and for my diagnostic clinic, just because of the resources, we still run qPCR on our assays because it's cheaper, and people have to pay for the service. And for the samples, you don't, they always send in palm samples where it's symptomatic, and when the palm's symptomatic, the phytoplasma is fully systemic, so you don't need that level of sensitivity that you get with the digital PCR. From a research perspective, there is a very specific niche that I use qPCR for, and that's with HRM analysis. If you're looking at a single SNP within a region or a few, you can't design assays that are specific for those and use it on the digital PCR to be exclusive. It's just not possible with every scenario. So that's a case where, like the insect species I work with that transmits the pathogen, we did a population genetic study in Florida and found four distinct haplotypes that differ by one to two nucleotides. Like really, really being stringent, you might be able to get it to where the probe doesn't work or one of the primers doesn't. But it's, you just can't get that specificity with some things that are so closely related, so with HRM, with the qPCR, basically we have that app that gives you really high resolution. So you know we run the qPCR, we amplify the CO1 gene for these bugs, and then eventually we want to use the HRM to be a cost-effective way to screen these genetic variants that only differ by one nucleotide.
Jordan Ruggieri 19:26
I mean, even just you walking through those different case scenarios, I think shows, right, like there's different ways and different techniques and different you know features and benefits of of each of the technologies. That again, just depending on what you're looking to do, you can use both or one over the other.
Lisa Crawford 19:43
Across so many applications and so many labs. The storyline is consistent. qPCR and dPCR aren't competing technologies; they're complementary.
Jordan Ruggieri 19:52
Today, we've heard great examples of how qPCR provides speed, flexibility, and scalability, and how dPCR provides. Precision, analytical sensitivity, and absolute quantification-both are valuable tools in a scientist's toolbox that can be used depending on the questions being asked, the data output needed, and the analytical sensitivity or specificity desired.
Lisa Crawford 20:15
Together, these techniques help researchers to see the full picture, starting with their first hypothesis to the final answer.
Jordan Ruggieri 20:22
With that, we wrap up another Absolute Gene-ius Science Snapshot. Stay tuned for more upcoming episodes and be sure to subscribe to Absolute Gene-ius to get the latest episodes as they are available. Until then, stay curious.
Lisa Crawford 20:35
This episode was produced by Sarah Briganti, Matt Ferris, and Matthew Stock. All products mentioned in this episode are for research use only, not for use in diagnostic procedures.
Jordan Ruggieri 20:44
Boom, baby.