To singleplex or multiplex?

Several key factors must be considered when designing a real-time PCR experiment, including how many targets you intend to detect. Many researchers opt for multiplex detection chemistries due to its ability to provide more data per sample with minimal added cost, but you need enough targets for it to make sense. When getting ready to start your real-time PCR experiment, one of the first key decisions is assay design, and you need to decide if singleplex or multiplex detection is the best fit. These two detection methods involve different assay design and setup, and each one has its own set of advantages and disadvantages.

Why multiplex

In multiplex PCR, multiple target sequences are amplified in a single reaction. In real-time PCR, these target sequences are commonly detected using probes that have different dye labels. Multiplex assays are attractive because they can help you save time, cost and most importantly, sample. However, multiplexing has its drawbacks. Primer design plays a crucial role in multiplex success, and often multiple rounds of optimization are needed to determine an appropriate primer concentration. Primers and unintended PCR products or artifacts may compete in amplification, resulting in uneven amplification for different targets. In fact, it’s common practice to perform singleplex PCR in order to amplify loci that multiplexing has failed to amplify.

Once optimized, multiplex assays can also be difficult to modify. Given the time and effort required to design a successful assay with a particular combination of compatible primer pairs, redesigning easily becomes a headache. With changes in routine assays inevitable to advance any system, whether for screening or testing, the ability to adapt quickly to these changes is not afforded with multiplexed reactions.

Why singleplex

Singleplex PCR is easier than multiplexing. Only one target is amplified per reaction, so your assay is easier to design and implement due to the absence of potential competition during PCR. However, many researchers find singleplexing limiting, since using several reactions to detect multiple targets can mean higher costs in materials and labor and more sample used per reaction.

The ability to scale an assay is also challenging when using singleplex reactions. Since each reaction must be isolated in an individual well, you can only scale to the number of reactions often done in 96- or 384-well plates that can be completed in a day. And when you consider the need for more reagents every time you add more reactions, cost can quickly become an issue.

Multiplex with singleplex simplicity with Standard BioTools microfluidic technology

Standard BioTools™ microfluidics technology assembles your PCR reactions for you in an automated, miniaturized fashion. Our integrated fluidic circuits (IFCs) have nanoscale reaction volumes, so you use less of your precious sample and reagent. Samples and assays are loaded into the IFC separately and then automatically combined in a pairwise manner within the closed system of the IFC to create individual singleplex reactions, with up to 192 singleplex reactions per sample (depending on IFC format). You can add, remove or replace assays on demand and scale throughput without changing technologies, allowing up to 96 individual samples and controls to be interrogated with up to 96 individual assays with the same dye label, for a total of up to 9,216 individual reactions per run. Think of it as multiplex throughput with singleplex simplicity.

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