Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency.

When developing multiplex Real-Time PCR assays, you need to consider primer design, the relative expression levels of target sequences, and master mix / reagent conditions.

Use the same design criteria for each primer/probe set and screen all sequences against each other to determine any potential primer-dimer formation. In addition, perform a BLAST analysis ( to determine primer specificity.

If the expression levels of the target sequences are significantly different, the most abundant target will be preferentially amplified and deplete all the reaction components, compromising amplification of the less abundant targets. One way to address this issue is to limit the primer concentrations of the most abundant target, using the lowest primer concentration that produces the same Cq and PCR efficiency. Limiting the primer allows the most abundant target to amplify and go to completion without depleting all the reagents needed for the other sequences.

Amplifying multiple target sequences creates additional demand for reaction components. Taq DNA polymerase, Mg++ and dNTP concentrations may need to be optimized to improve amplification of all targets. Master mixes optimized specifically for multiplex Real-Time PCR are now commercially available, and can reduce the amount of time required for optimization.

Compare experiment data that uses a single assay with the experiment data when the assay is combined into a multiplex assay. Sensitivity and PCR efficiency needs to be the same in both types of assay use.