PCR multiplexing enables scientists to measure multiple DNA or RNA targets in a single reaction, unlocking more insight from limited samples. This episode revisits real-world examples showing how multiplexing expands experimental design, improves precision, and simplifies complex workflows.
Multiplexing is reshaping how scientists think about PCR experiments, turning single-target workflows into powerful, multi-dimensional analyses.
In this Science Snapshot, we revisit standout moments from past episodes to explore how multiplexing is applied across research areas, from measuring interferon-related genes in lupus to analyzing complex microbial communities and detecting low-level donor-derived DNA.
Guests highlight both the opportunities and challenges, including assay design, probe optimization, and troubleshooting strategies. The episode also showcases how multiplexing enables entirely new applications, such as comparing chromosome ratios, assessing molecular integrity, and distinguishing functional mRNA from truncated byproducts. Whether using qPCR or digital PCR, multiplexing allows researchers to extract more information per sample, improve precision through multiple data points, and better reflect biological complexity. As platforms and chemistries continue to evolve, multiplexing is becoming more accessible, helping scientists move faster, conserve material, and focus more on interpreting results rather than optimizing reactions.
Jordan Ruggieri 00:00
These phone booths, guys, it's this picture of Superman that's on the side that says, "Share this phone booth. Get out after an hour." So it doesn't quite make sense, because Superman gets in and changes real quick. And their whole point is for you to get out faster. So it should be, "You should be like Superman, get in and get out.”
Lisa Crawford 00:30
Welcome back to another Science Snapshot, part of the Absolute Gene-ius podcast series from Thermo Fisher Scientific I'm Lisa Crawford.
Jordan Ruggieri 00:38
And I'm Jordan Ruggieri. Today, we're taking a closer look at PCR multiplexing, a strategy that allows scientists to measure multiple DNA or RNA targets within a single reaction. Rather than testing one gene at a time, multiplexing brings several targets into the same well using different fluorescent probes to distinguish them, the result is more data from less sample and often deeper insight into what's really happening biologically.
Lisa Crawford 01:03
Throughout this series, we've heard how multiplexing helps researchers move faster, conserve precious material and in some cases, examine molecular relationships that single target assays simply can't reveal.
Jordan Ruggieri 01:13
So today, we're revisiting some of our favorite guest moments that show how multiplexing, whether in qPCR or digital PCR, can improve precision, expand experimental design and support research workflows that grow with the complexity of the science.
Lisa Crawford 01:27
Let's start with a straightforward example of multiplexing in action. In season three, Arianna Arbona described how her team used digital PCR to measure four interferon related genes in lupus samples, all within a single well. By designing a 4-plex assay, they were able to generate more information from each precious sample, helping them understand disease biology more efficiently. Here is Arianna.
Arianna Arbona 01:50
We were studying lupus. So the thing would be that we were trying to see in a multiplexed digital PCR. So we designed with Thermo Fisher, the TaqMan probes, and we had four different genes that we were trying to see in those samples we have from lupus. So we designed the probes in a way we could measure four genes at the same time in one sample. So the idea would be to have a faster and easier way to see if you already know, like you already have a sample, and you want to test the levels of three, four different genes regarding and that we already know they're affected in those in that disease. So then you can, just with a digital PCR, have the levels of the four of them at the same time.
Jordan Ruggieri 02:49
Measuring four genes at once sounds simple, Lisa, but we know that behind the scenes multiplexing requires careful assay design.
Lisa Crawford 02:57
That is true. When multiple primers and probes share the same reaction, small design decisions can make a big difference from sequence specificity to fluorophore choice. In season two, Ronak Feiglman and Kimi Soohoo Ong gave us a closer look at how they approach multiplex design and how they troubleshoot when assays don't behave as expected. Let's listen.
Rounak Feigelman, PhD 03:17
So first of all, like when we are actually designing the primers and probes we like to see like, you know, if we are avoiding any natural variation, population-wide variation, while we are like, you know, designing our primers and probes. And after that, basically, we try to make sure that our primers and probes do not, like you know, form primers and dimers amongst themselves and with, within themselves also. After that, we also like to check for specificity in the genome in terms of targets. Sometimes, like you know, if your targets are close to one another, then we need to be more creative in our designs, because we do not want, like, competition between our probes. There, like, you know, some more advanced methodologies come into play.
Kimi Soohoo Ong 04:17
I was going to add after, I guess, we have an assay designed and it's tested and then we need to do some troubleshooting. Like, some different things we'll play with is, like, probe length, or like, if it's like a SNP. So there's two probes we'll play with, like, the way, like, either the probes are, like, right on top of each other, or we'll like, stagger the start and stops. And then sometimes we'll also play with the quencher, so we have MGB or QSY quenchers. And then occasionally we'll also switch the strand of the probe, so from like the positive strand to the negative strand, just to try different ways of troubleshooting.
Rounak Feigelman, PhD 04:57
Even if you design an assay for qPCR, you can try running it on digital PCR and see whether it performs well. Lot of times it will perform well, but there are some considerations for digital PCR. Digital PCR the readout is at the end point, 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 end point. 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 05:46
Up to this point, we've talked about combining a few targets in a single reaction, but multiplexing doesn't stop there.
Lisa Crawford 05:52
In some situations, researchers are looking to measure far more complexity, where even a handful of targets isn't enough.
Jordan Ruggieri 05:58
That was the case in season one, where Ray Ketchum explained why digital PCR became central to his quality control strategy when working with a 22 species bacterial consortium. Here's Ray.
Ray Ketchum, PhD 06:09
We wouldn't be able to do what we're doing without digital PCR. The reason for that is because we have a product that has 22 different bacteria together. It's impossible to plate these bacteria and count them, you know, all at once. Some bacteria don't plate very well. Some of them are anaerobes. Some of them are aerobes. So they can't grow under the same conditions. So all of these variables make digital PCR really a keystone to the technology and to our quality control. So what we do is we do a whole genome sequence for each of our bacteria. We figure out within that genome which genes are single copy and which ones are unique to that particular species, and then we design primers and probes for that portion of that particular gene. Or in some cases, it's just a DNA sequence. It may not actually be a coding gene. And then, once we have that, we can take a very complex mixture of bacteria, do a total DNA extract on that, and then analyze that. Again, with the assumption being that every signal that we get on digital PCR relates back to one single bacterium. And by that we can actually get some, some pretty good, pretty good numbers.
Lisa Crawford 07:26
Nice example where multiplexing makes it possible to measure complex mixtures, even when organisms can't be cultured together.
Jordan Ruggieri 07:33
Yeah, but complexity isn't only about the number of organisms. Sometimes it's about detecting very subtle biological signals within a highly diluted sample.
Lisa Crawford 07:42
In this clip from season two, Dr. Leanne Baxter-Lowe shared how her team developed a high-plex digital PCR approach to measure donor derived cell-free DNA, detecting up to 12 targets in a single well. Let's listen.
Lee Ann Baxter-Lowe, PhD 07:54
That's exactly what we've been working on is multiplexing and developing primers and probes that can be used to differentiate donor and recipient. We are using indels, insertions and deletions, and the alleles that we're using now have been selected so that they're between point five or point six frequency in the population. The goal there is to have as many informative markers as we can. And we started by trying to develop a whole new approach to digital PCR that allows us to multiplex beyond what's been available before. We've recently published a paper in Frontiers in Genetics, describing this approach in detail. Interestingly, what we did in in the first demonstration is that we could detect 12 different targets in one well, instead of one. It would be very difficult to do, but we think it could be done that you could get up to 25 targets per well. And then you know, if you, if you use, you know, multiple wells for one sample, then you can also increase the number of targets beyond that. But in measuring donor derived DNA, it's my observation that having many targets and being able to average them is beneficial because you're working at something that's just so, such a low level, and it improves precision and accuracy when you have multiple informative targets.
Jordan Ruggieri 09:40
And going even further, multiplexing isn't just about more targets, it's also about comparing them directly.
Lisa Crawford 09:46
Parker Wilson used a multiplex digital PCR assay to measure X and Y chromosomes in the same well. Here's Parker.
Parker Wilson, MD PhD 09:53
This is great for digital PCR, and some digital PCR approaches came out in the last couple years. And people have published on this to look at loss of Y chromosome in blood. And conceptually, this is very easy to think of, you just design primers and probes for different chromosome targets. The nice thing about the assays that we use is you can use a multiplex reaction. You can design multiple primer probe combinations to estimate a ratio of the relative number of chromosomes within the tissue. So what we did for this paper we published back in January, is we designed a digital PCR assay where we have one primer probe set as targeting the Y chromosome, another is targeting the X chromosome, and then we estimate the ratio between the quantity of X and Y chromosomes in the tissue.
Lisa Crawford 10:55
Counting and comparing targets is powerful, but multiplexing can go beyond quantification, right.
Jordan Ruggieri 10:59
Right again. Instead of simply measuring abundance, researchers use multiplexing to evaluate molecular integrity, to tell if multiple targets are connected on the same molecule. Here is Marcia Slater describing how multiplexing in dPCR makes that possible.
Marcia Slater 11:16
There's a new kind of up-and-coming application that people will refer to as molecule integrity. So, it's something that you actually can't even do with qPCR. So you may have noticed that earlier I said that if you want to be totally technically correct, that digital PCR measures molecules. So for most applications, molecules and copies are the same things. But imagine if you had a construct, and you not only wanted to know how much of that construct you had, but is the construct intact versus getting degraded? So with the machines today, you can do a multicolor analysis, they're enabled for high levels of multiplexing. So you can put a different color probe assay spanning that molecule of interest, and then the question you can ask is, are all four colors together in my sub reaction, in my chamber? If they're together, then that kind of implies that they were on the same molecule. Think of those as being like tied together with a rope and they're all being pulled together into the same micro chamber. If instead, you only have a subset of colors, so if you did a four color one and only two are there, well then that molecule is not there in its entirety. It must have been cut apart, otherwise all four colors would be there. So digital PCR really allows this kind of up-and-coming application to see if what you're doing is intact or if it's been degraded.
Jordan Ruggieri 12:51
That's an oldie but a goodie.
Lisa Crawford 12:53
Another oldie but goodie takes this concept even further. In season two, Christian Cobaugh described how his team uses digital PCR to better distinguish active mRNA from shorter synthesis byproducts. Here's Christian.
Christian Cobaugh, PhD 13:06
We're very excited about using dPCR to quantify the active mRNA. One of the issues with RNA synthesis is you get a lot of truncation products. It could only be, may only be 5%, could only be 10% and for a lot of folks, that's considered a success. But the current analytical methods don't do a great job sorting those out as separate from the main product. If we design dPCR assays to land on the right spot of the mRNA, we get a lot closer to the truth in terms of what is the active material. This is not something you're going to find in the USP, and I'm not sure you're going to find any other company that's doing this. And so we're super excited about that because, I mean, we've got, again, some orthogonal data, including sequencing data, that's showing us that we're heading in the right direction. We still need to move this into a qualified environment. But, you know, this is very differentiated, and honestly, I'm not sure of any other technology that can be that accurate in its ability to evaluate the active component of your DS in an mRNA medicine.
Christina Bouwens 14:31
And if I can ask this is probably tied to the ability to not just look at total length, but the ability to interrogate single molecules in their state.
Christian Cobaugh, PhD 14:40
Correct. Yep. And you know, you can tile assays because it's you can multiplex. Eventually we see a way to do this with just a single assay on the on the RNA. So that that multiplexing, right now is there to kind of develop the method, but ultimately, when we go to qualify this as a release assay, we think this will be a singleplex approach.
Jordan Ruggieri 15:08
Okay, we've heard how multiplexing can tackle everything from microbial communities to mRNA integrity, but in day-to-day lab work, sometimes the goal is simpler.
Lisa Crawford 15:17
Like just getting reliable data without extensive troubleshooting. Here's Patrick Hannington explaining how multiplexing became more approachable in his lab.
Patrick Hanington, PhD 15:25
In terms of multiplexing, we always struggle, A with the presence or absence of inhibitors, but then B like, part of the part of our challenge sometimes, is that we're, we'll be working with assays that that can be, you know, depending on the fluorophore depending on what we're amplifying, can cause challenges when we multiplex. And so we've, we've often defaulted to just always running a single fluorophore assay with the qPCR. It's not that we can't multiplex on the qPCR platforms, but there's a lot more that we have to do in terms of compensation and figuring out exactly, you know, how the two different assays perform with each other. The thing that we found with the Absolute Q is that it's essentially set up so that the three, three of the fluorophores for sure, are like very distinct, and then the fourth one is usually like, also pretty good. And so we find that it's a lot easier to just run those four and we can, we can confirm that there's no bleed over a lot easier with that software. So it's just a very simple multiplexing platform. And because of the simplicity of the way that the system works, generally, it's just really easy for us to just load it up and run the samples with four different fluorophores in there and then get the data we need from all four. With the qPCR, we often found that we had to, we had to work on optimizing enzyme amount versus DNA that we loaded into the reaction versus the presence of like all the different components of the reaction and the Absolute Q system just works a lot easier to do that multiplexing.
Jordan Ruggieri 17:00
Across all of these stories, multiplexing shows up in different ways, expanding experiments, improving precision and helping researchers ask more nuanced questions.
Lisa Crawford 17:09
From four genes in a Lupus sample, to multi species communities, chromosomal ratios, and RNA integrity, multiplexing really adapts to the scale and complexity of the science.
Jordan Ruggieri 17:14
And I feel like I've got to say that when PCR platforms are designed to simplify the process, that capability becomes even more accessible, allowing scientists to focus on interpretation rather than optimization.
Lisa Crawford 17:32
You mean like Applied Biosystems PCR platforms, Jordan?
Jordan Ruggieri 17:36
Absolutely. Curious about multiplexing and how you can get more data from a sample. Check out the TaqMan™ multiplex solutions from Thermo Fisher Scientific.
Lisa Crawford 17:45
Whether you're facing challenges like signal crosstalk, poor amplification or channel limitations, our comprehensive solutions are designed to help you achieve cleaner curves, consistent results and efficient detection of multiple targets in a single reaction. TaqMan multiplex solutions help make complex qPCR and dPCR experiments simpler, cleaner and more efficient.
Jordan Ruggieri 18:06
With optimized master mixes, TaqMan probes supporting up to six targets, and instruments plus analysis software built to work together, you can save precious sample, cut reagent costs, and get consistent, reliable results.
Lisa Crawford 18:19
Also be sure to check out the multiplex optimization guide, providing step by step tips and tricks to help get the results you need.
Jordan Ruggieri 18:26
To explore TaqMan probes and access the multiplex optimization guide, visit thermofisher.com/multiplexqpcr. That's thermofisher.com/multiplexqpcr.
Lisa Crawford 18:38
Thanks for joining us for today's episode of Science Snapshot. We hope these stories inspire new ideas in your own research. Stay curious, and we'll see you next time on Absolute Gene-ius. This episode was produced by Sarah Briganti, Matt Ferris and Matthew Stock.
Jordan Ruggieri 18:53
Products mentioned in this episode are for Research Use Only, not for use in diagnostic procedures.