Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
If real-time qPCR is good for measuring gene expression and target abundance, multiplex qPCR is even better, right? You can use the same amount of sample, save on time and reagents, get better data and answer multiple questions—what’s not to love?
Multiplex qPCR is a common tool for single nucleotide polymorphism (SNP) detection, gene expression and other applications for which a large number of samples, or precious samples, need to be screened for a small number of targets. But multiplex qPCR isn’t as simple as it sounds, with the complexity of designing and validating the assay increasing with each new target.
Here we look at some of the benefits and pitfalls of combining qPCR assays into a single tube and share some tips to help with the process.
Double down
Perhaps the most common application of multiplex qPCR is also the simplest, combining reactions for the target of interest and a reference gene in the same tube, for instance for high-throughput genotyping or gene-expression analyses. “There are benefits to that—reducing the workflow time, the cost and increasing data precision because you’re not pipetting the sample into multiple wells,” says Paul Streng, global product manager for gene expression at Bio-Rad Laboratories.
Given obvious design criteria, like matching annealing temperatures and ensuring the reactions don’t differ dramatically in Cq value (the number of cycles it takes to reach a given level of fluorescence), turning two pre-validated simplex assays into a duplex reaction is “pretty much plug and play,” Streng says.
The more the merrier?
For higher levels of plexing, in the bacteriology or virology space, for instance, things become decidedly more complicated.
Why? Combinatorial complexity. Whenever components are mixed in a tube, there is always the potential for them to react with each other in unexpected ways. The more components, the greater is the potential. The problem for multiplex qPCR is that individual reactions may be impacted to different degrees.
For instance, when doing mutation or SNP detection, using probes that may differ by only one base, “if the DNA contains any material that will inhibit the reaction, then you can have problems discriminating [between the two sequences],” warns Tania Nolan, global manager for applications and technical support at Sigma-Aldrich. Nolan has seen heme carryover enhance one fluorescent dye while quenching another, for example. “The quality of the template makes an enormous difference.”
Beyond starting with good templates, initial experimental design has the greatest influence on qPCR, says Nolan, so researchers should try to design their assays as multiplex panels from the start.
Nolan relies on Beacon Designer™ (list price US$2,885) from Premier Biosoft, which can devise multiplex reactions for up to five genes in a single tube, in the process checking probes and primers for cross compatibility (to eliminate primer dimers, for example) and avoiding cross homology to other targets. The software first designs assays for each target and then checks for those that can be run together.
Many assay design tools are available, including a free version of Beacon Designer and Sigma-Aldrich’s OligoArchitect™ that was “built by the same people,” says Nolan. These tools accomplish most of the same tasks as Beacon Designer, “but you have to do more manual work yourself. Beacon Designer is a slicker way of doing it.”
After the multiplex qPCR assays are designed, it’s important to optimize and validate each reaction individually before putting them together. (Using pre-validated or pre-formulated assays such as Bio-Rad’s PrimePCR™ or Life Technologies’ TaqMan® offerings, for example, can certainly help.) Then make sure the reactions perform equivalently in multiplex. It’s normal to lose dynamic range in the latter, and the Cq values for each target may change, but the relative differences in Cq between the different assays should remain consistent in multiplex.
Optimizing for qPCR
PCR requires more than just good primers, however. There are a host of master mixes on the market either specifically designed for, or optimized to work in, multiplex reactions. “Because you have more reactions going, you may need a little more enzyme; you may need a little more dNTP—these are the sorts of things we can support with [a] multiplexing master mix,” says Sundiep Phanse, director of product management at Life Technologies, a brand of Thermo Fisher Scientific.
Vendors use different, sometimes proprietary formulations that work with different enzymes and chemistries. Some of Life Technologies’ master mixes, for example, include uracil DNA glycosylase (UDG) to make it “a little more contamination-tolerant” and a passive reference dye to normalize for signal variation, says Phanse.
Multiplex qPCR almost invariably makes use of sequence-specific probes (rather than double-stranded DNA dyes like SYBR that indiscriminately pick up all PCR products). These are typically composed of a fluorophore and quencher system, and they must be spectrally matched to work with each other as well as with the instrument on which the reaction will be run. But just matching the dyes isn’t enough: It’s also best to use the brightest dye with the lowest-expressing target and the dimmest dye with the most abundant target.
Sometimes it’s necessary to empirically tweak the reactions, perhaps by changing the Mg+ concentration or adding more dNTPs, although caution should be used to avoid throwing off the balance of the chemistry. If the expression levels of the individual reactions are too far apart, reagents may be used up by the more abundant target before the less abundant molecules can even be detected. In that case, Streng suggests limiting the amount of primer so the fast expressor will plateau early.
For more difficult targets, like rare somatic mutations, it may be necessary to look to alternate probe chemistry such as Scorpions® or Molecular Beacons. It’s also possible to incorporate locked nucleic acids (LNAs) to generate shorter probes and influence melting temperature, which “helps to destabilize the mismatch,” says Nolan.
Multiplexing qPCR reactions beyond duplex is not something to be undertaken casually. It takes time and resources to optimize, and it may not be worth it for someone only running a few samples or frequently changing targets. But after the assay is up and running, you have a screen that can save time, resources and sample for years to come.