Multiplex qPCR is a technique that simultaneously amplifies two or more target sequences in a single reaction. Analysis of multiple targets per sample allows for conservation of limited starting materials, the ability to run an internal control (i.e. a housekeeping gene in a gene expression assay), and increased sample throughput. Multiplexing is generally more complicated than simply adding additional primers and probes to a singleplex qPCR. Additional consideration must be given to the selection of primers, probes, cycling conditions, and reaction formulation.
Figure 1.Example of Dynamic Range Loss for NON-Optimal Multiplex qPCR
In multiplex qPCR, as in a singleplex qPCR, it is critical to start with quality primer and probe sets. Each set used in a multiplex reaction should be designed and evaluated for optimum reaction metrics. Quality primers can be designed by using one of several commercially available software programs. Sigma-Genosys utilizes Premier Biosoft Beacon Designerâ¢ in its probe assay development service. A freeware alternative for primer design is Primer3. For multiplex qPCR, it is suggested that primers with nearly identical melting temperatures (Tm), 45-60% GC content, and no sequence homology with other primers be used.1 After primer design, empirical testing of each primer set is recommended. Metrics to consider when evaluating an individual primer set are: single target specificity, qPCR efficiency, and dynamic range. To confirm that each selected primer set amplifies one product, perform qPCR and assay by agarose gel electrophoresis.
Optimal individual primer sets are a key foundation to multiplex qPCR as individual amplicon dynamic ranges and efficiencies may deteriorate when combined into a multiplex reaction. An example of dynamic range deterioration from 6 to 3 logs is shown in Figure 1. Efficiency is a measure of the amplification yield after each PCR cycle and must be determined empirically. Efficiency and dynamic range are derived by plotting the log of known template concentration vs. Ct. The slope of the standard curve is proportional to the efficiency with â3.32 being 100% efficient. In practice, a slope between â3.50 and â3.14 is typically acceptable. The dynamic range is generally described as the range of concentrations for which the slope of the standard curve remains linear with an R2 value greater than 0.98. The best multiplex reactions will be achieved using primers that have high individual efficiencies and broad dynamic ranges. Primer/probe sets affording the same efficiency in individual and multiplex reactions are highly desirable for multiplex qPCR. Often it is necessary to design and evaluate several primer/probe sets before finding the best set for a particular target.2,3
In addition to carefully selecting the primer/probe sequences, it is important to choose a combination of fluorophores that has compatible excitation and emission spectra. Your real-time qPCR instrumentâs capabilities (filters) are critical to keep in mind when selecting fluorophore combinations for multiplexing.
The nucleotides, enzyme, and buffer concentrations in Sigmaâs JumpStart Taq ReadyMix for Quantitative PCR, Catalog Number D7440, have been optimized for a wide variety of singleplex qPCR applications. To tailor the mix for multiplex qPCR, an elevated magnesium level is needed. A final concentration of 6 mM Mg2+ is appropriate for most multiplex reactions. Figure 2 demonstrates the improved sensitivity from supplementing magnesium levels above the standard 1.5 mM. In this example, some multiplex products were not even detected using 1.5 mM MgCl2.
Magnesium supplementation can be achieved by following the instructions in the JumpStart Taq ReadyMix for Quantitative PCR Technical Bulletin, Catalog Number D7440. For a 20 Î¼L multiplex reaction use the standard 10 Î¼L of JumpStart Taq ReadyMix, Catalog Number P2893, primers, probe, template, reference dye (if necessary) and add 3.6 Î¼L of the provided 25 mM MgCl2, Catalog Number M8787. Mix and place in a thermocycler. In some cases, it may be necessary to increase the magnesium level even further. Typically the optimum working level is between 5 and 20 mM. Should reaction volume become a concern, use 1 M MgCl2, Catalog Number M1028, diluted to 100 mM in place of the 25 mM MgCl2.
If the magnesium level has changed since primer specificity was verified, re-verification of specificity with the final reaction conditions is suggested. If additional sensitivity is required after MgCl2 optimization, then decreasing primer concentrations should be tested. Primer concentrations at low as 0.01ÂµM can result in improved detection.
Figure 2.Effect of MgCl2 on Multiplex qPCR
An accurate comparison between multiplex targets is dependent on all targets in the reaction having similar efficiencies. After the initial primer/probe set selection, multiplex qPCR optimization should focus on optimizing the target product reaction efficiencies, including specificity. One way to do this is to increase the primer concentration for the poorly amplified target, but one must be mindful that non-specific amplicons are also more likely to be generated. The most robust and accurate multiplex will have absolute specificity across the testing range and efficiencies near theoretical.
Increasing other reagent concentrations such as polymerase, dNTPs or salts usually do not result in significant differences in PCR efficiency, although there are exceptions. One caution is that increased dNTP concentrations will require increased magnesium, as the polymerase uses these substrates as a dNTPâ ¢Mg complex. A free magnesium concentration of 0.5-1.0 mM is necessary to support PCR. Levels of dNTP that exceed Mg2+ will poison the reaction.
Specific reactions with unequal reaction efficiencies can be accommodated in two ways. The best way is to generate a standard curve for each target under multiplex conditions and mathematically correct for the reduced PCR amplification. The alternative is to make all primer/probe set efficiencies equal by decreasing the relative primer/probe set concentrations of the efficient reaction(s).1,3-5 This latter course should only be used as a last resort, as it reduces the sensitivity and linear range of the multiplex assay.
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No license is conveyed with the purchase of this product under any of US Patents Nos. 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the United States, or any other patents or patent applications, relating to the 5â Nuclease and dsDNA-Binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.