Multiplexing Pros and Cons
How, When, and Whether or Not to Multiplex Your qPCR Experiment
One of the most common questions when designing a quantitative PCR (qPCR) experiment is whether it’s possible to amplify multiple target sequences in a single reaction. This is called multiplexing, and good news – it is indeed possible. However, it does present some challenges that must be addressed to make sure your experiment is successful. In episodes 22 and 23 of the Taq Talk video series, we review some of the need-to-know insights into how, when, and whether or not to multiplex your qPCR experiment.
Compare and Contrast
Singleplexing
First off, singleplexing (amplifying a single target per reaction) is much simpler than multiplexing. Real-time PCR protocols are usually built around singleplex experiments, so switching to multiplexing requires adjustments to the protocol. Singleplexing also comes with a lack of ambiguity, meaning that when only one target sequence is amplified per reaction, there’s no possibility of different amplification events interfering with or combining with each other to complicate readings.
Multiplexing
Multiplexing offers some important advantages that definitely make it worth considering. When multiple target sequences are amplified in a single reaction, there is a substantial savings in master mix reagents. Half as many wells or fewer means half as much total dye, dNTPs, and more to acquire and spend – and that cost savings is appealing to many laboratories.
Additionally, pipetting issues sometimes mean that the total reaction volume isn’t exactly the same between all qPCR reactions. This can be a problem when, for example, comparing a target gene to a reference gene analyzed in separate singleplex reactions.
In a multiplex experiment, multiple genes are analyzed in a single reaction, so the volume between targets must be the same. With fewer wells to fill, setting up a multiplex experiment is also much faster than setting up an equal number of singleplex reactions.
However, since qPCR should generally be run using identical triplicate reactions, even singleplex experiments have a built-in way to monitor pipetting precision. For this reason, multiplexing is rarely, if ever, mandatory, but it can still represent a sizable savings in reagents and time if its complexities are managed.
Related video: Considerations for Singleplex vs. Multiplex qPCR – Taq Talk Episode 23
Optimizing a Multiplex qPCR experiment
Target Sequence Requirements
Getting good data out of a multiplex experiment takes special care. Each target sequence needs its own dye and associated probe with a unique emission wavelength that your qPCR machine can detect and distinguish. Dye choice is also very important: Applied Biosystems™ SYBR™ Green master mixes are designed for singleplex reactions only, because SYBR Green dye binds to all PCR products. With Applied Biosystems™ TaqMan™ Assays, the individual targets are easily distinguished using probes labeled with different fluorophores, such as FAM™ and VIC™ dyes. Any Applied Biosystems™ real-time PCR instrument can distinguish between these dyes.
But wait – simply putting all of that together in a single reaction might not be enough to get good data. When two reactions share a well, even when they have different probes, they compete for the dNTPs, enzymes, and other reaction components (Figure 1).
If one gene (usually the endogenous control) begins amplifying before the other does, it may hit its linear and plateau phases before the other even shows up on the amplification plot. The target gene then ends up starved of reagents, leading to poor amplification and results that cannot be interpreted (Figure 2).
Figure 1. Competition for reaction components in a real-time PCR assay. Reaction wells (in triplicate) are represented in the center.
Figure 2. Early amplification of endogenous control resulting in poor amplification of target gene.
Related video: How to Optimize Multiplex qPCR Experiments – Taq Talk Episode 22
You’re in luck – avoiding this problem can be surprisingly simple. If one target is likely to outcompete the other for reagents, that target can be primer-limited. Significantly reducing the primer concentration for the control gene causes it to plateau much earlier, making the two signals easier to distinguish and preserving the other reagents for the experimental gene of interest.
Multiplexing Two Genes
Sorting out how easy it will be to multiplex two genes, which one to primer-limit, and whether two dyes are truly multiplex-compatible requires some test samples. Start by selecting 5 – 6 samples each from your experimental group and from your control group and run them all in both a duplex and a singleplex configuration. If the results are comparable, such that there is little difference in the readings between the two configurations for most samples, then it should be safe to run the rest of the experiment in multiplex. If the two readings do not agree, it’s likely that running the experiment in multiplex will lead to inconsistent or even unusable data. If samples are in short supply or if sample preparation takes a substantial amount of time, avoiding the risk may be preferable.
Higher-Order Multiplexing
Similar caution should be used in higher-order multiplexing. Applied Biosystems™ real-time PCR instruments are equipped with multiple filter sets that allow you to multiplex numerous primers and probes in a single reaction. However, the more targets there are in a single reaction, the more complex the interactions between them will be and the greater the danger of the results being skewed in some way.
Applied Biosystems™ TaqMan™ Assays use robust, highly specific, and exceedingly flexible real-time PCR technology. Users have the option to select TaqMan probes with different dyes, which allows them to confidently and successfully multiplex with more than one target in a single well.
Learn More
» Real-Time PCR Digital Handbook 2.0 – Discover real-time PCR best practices, basic principles, and troubleshooting tips.
Applied Biosystems™ Taq Talk Video Series
» Check out the entire Applied Biosystems Taq Talk Video Series – As experienced professionals in real-time PCR, we address your most frequently asked real-time PCR questions and challenges in this new video series. Browse the lineup and stay tuned for more Taq Talk videos.
Watch the Videos Referenced in This Article
» How to Optimize Multiplex qPCR Experiments – Taq Talk Episode 22
» Considerations for Singleplex vs. Multiplex qPCR – Taq Talk Episode 23
—
For Research Use Only. Not for use in diagnostic procedures.
© 2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license
To whom it may concern,
Can I please kindly know whether there’s any possibility to perform multiplex qPCR for the following genes of Proteus mirabilis. There are predesigned gene expression assays in single tubes.
MrpA Gene
UreC Gene
SpeA Gene
RplS Gene
RsbA Gene