Are You Making the Most of Your Multiplex qPCR?

Multiplex qPCR: that’s just running several qPCRs at once, right? In essence, yes—multiple targets are amplified within a single tube. But when used correctly, the benefits of this technique over single-plex qPCR can be substantial. You can minimize the amount of starting material required, something which can be extremely beneficial when sample sources are limited. Even when there is an abundance of starting material, multiplexing can increase throughput and decrease sample handling, saving on the cost of reagents and other consumables.

Is your multiplex assay providing you with the best possible results?

Primer and probe design are essential components to the success of any PCR experiment. For multiplex qPCR, multiple primers and probes must be designed to have compatible Tm values, and sequences that do not interact. An online library of predesigned assays for human, mouse, and rat target genomes is available from IDT. They have been individually BLAST searched across the transcriptome, reducing cross-reactivity. If you are working with a different species, there is another assay selection tool available.

What should you consider when setting up your multiplex qPCR?

As with any qPCR assay, design of a multiplex experiment requires some optimization. Amplification of target sequences can be affected by factors such as gene expression levels, primer interactions, and competition for reaction reagents. The following tips will help ensure successful experiments.

  1. Check for primer and probe sequence interactions

    If you are designing your own assays, it is critical to use a design tool with a validated algorithm. IDT offers online software to identify primer/probe hairpins, homo- or heterodimers, and primer/probe complementarity across the different assays. You should also avoid extensive 3’ overlap (>4 bases) between primers to reduce the incidence of nonproductive amplification.

  2. Use an appropriate and unique reporter dye to identify each target

    It is important to determine the dyes for which your qPCR instrument has been calibrated. Then you can select from those the dyes with appropriate excitation wavelengths, and that have little or no overlap in emission spectra. Match high-intensity dyes with probes designed to detect low abundance transcripts.

  3. Minimize signal cross-talk by using probes that quench well

    Highly efficient dark quenchers, especially those used in combination with a secondary quencher, have an added advantage. They reduce background fluorescence considerably and increase sensitivity and accuracy of resulting data.

  4. Optimize the individual reactions and validate the multiplex reaction

    Each reaction needs to be >90% efficient. You can test this by running individual assays, even for assays that have been published or successfully performed in other labs.

  5. Validate the multiplex reaction

    You should run each assay in a single-plex experiment before multiplexing to confirm that the Cq values are accurate, and not artificially reduced by once multiplexed. Run a combined reaction alongside the individual reactions to provide a measure for comparability.

  6. Optimize the multiplex reactions

    The number of targets, target abundance, and amplicon length will all affect the rate of reagent consumption. If any reaction components are limiting, there can be significant delays in Cq or a total loss of PCR product. Master mixes can be purchased that are specifically formulated for multiplexing. If you plan to use a single-plex master mix, you must adjust the concentration of reaction components. Another point to note is that the standard 2:1 primer:probe ratio for each of the genes analyzed may need to be adjusted to achieve optimal results.

Ellen Prediger is Director, Scientific Communication, IDT, Coralville, IA, U.S.A.;