In multiplex PCR, multiple targets are amplified in a single reaction tube. Each target is amplified by a different set of primers and a uniquely-labeled probe that will distinguish each PCR amplicon. Multiplexing provides some advantages over single-reaction PCR, including a lower amount of starting material, increased throughput, lower reagent costs, and less sample handling. However, the experimental design for multiplexing is more complicated as the amplification of each target can affect others in the same reaction.
Therefore, careful consideration of design and optimization of the reactions is critical. IDT PrimeTime® Predesigned qPCR Assays and PrimeTime Custom qPCR Assays offer multiple dye/quencher combinations and primer/probe ratios to simplify the multiplex experiment design.
Reporter dye choice
Each target must be identified by a separate reporter dye. Select dyes so that fluorophore emission spectra overlap as little as possible, noting that some instruments are compatible with only certain dyes—check the documentation for your instrument to be sure the dyes are compatible. It is a good idea to select FAM for low copy messages for its strong signal. Lower-signal fluorophores can then be used for the higher abundant messages. IDT PrimeTime qPCR Assays come with 5 different dye/quencher combinations for the best fit for your instrument and multiplexing needs. See articles in the Additional resources sidebar (top right) for recommendations for dye and reporter combinations for different PCR platforms.
During multiplex PCR, master mix reagents can be depleted fairly quickly for highly expressed genes due to the limited amount of reaction components. For these assays, it is preferable to limit the primer concentration of higher expressing genes. If the primers are limited, the amplification of the higher expressing genes will decrease, thereby making the mastermix ingredients more available for amplification of lower expressing targets.
The primer-to-probe ratios that work best will depend on your particular experiments, but a good starting point is to limit the primer-to-probe ratio for the highest expressing genes to 1:1 and increase the ratio for the lowest expressing genes to 4:1. IDT PrimeTime qPCR Assays allow you to select a premixed primer/probe ratio from 1:1 to 4:1 to maximize experimental flexibility.
Validate the multiplex reactions by running a multiplex reaction in parallel with an individual reaction to make sure that similar Cq values are obtained. Compare the standard curves and verify that the Cq values are similar at both the high and low ends. A good multiplex will have similar curves and similar limits of detection.