Considerations with multiplexing

Three key points to keep in mind

To help you get into the multiplexing workflow efficiently, there are a few key points to consider:

  • Firstly, regarding primer design, researchers should make sure to validate new primers separately in a singleplex reaction to ensure they work efficiently. One should also keep in mind that primers should not have more than a 3°C difference in Tm. Primers should be experimentally checked for primer dimer formation.
  • When it comes to probe design, researchers should make sure that each probe has a fluorescent label that is detectable in a different channel. One must also ensure probe label and quencher compatibility. With quenchers, it is important to note that some of them can change probe Tm significantly. Probe Tm should be 7-8°C higher than primer Tm.
  • Finally, it is important to address qPCR cycler limitations. Namely, the number of targets per reaction is limited by the number of channels in the cycler. It must also be considered that in some systems one channel is reserved for signal normalization by using passive reference dyes such as ROX or Mustang Purple® (or their analogs), and the same channel cannot be used for normalization as well as target detection. Calibration requirements should be followed according to the manufacturer's guidelines.

Multiplexing Solis BioDyne

Figure 1. SolisFAST® Probe qPCR Mix (ROX) was used in a 4-plex probe-based qPCR with fourteen fold serial dilutions of human gDNA (40 ng –40 pg per reaction, three replicates at each concentration). qPCR was performed on a QuantStudio™ 6 Flex qPCR cycler (Applied BioSystems™) using ROX dye for normalization.

Choosing the right tools

Using regular qPCR mixes in highly multiplexed assays carries a number of risks, such as late Ct values, low efficiency, low sensitivity, and low fluorescence. As regular qPCR mixes do not have a sufficient amount of components for amplifying more than 2 targets simultaneously, you are running the risk of getting false-negative results. Using specifically designed multiplex mixes will greatly increase your success.

Solis BioDyne multiplex-ready probe-based qPCR Master Mixes:

For fast cycling speed and up to 5-plex reactions:

For standard cycling speed and up to 4-plex reactions:

By using the right product designed for multiplexing, you can rest assured you will enjoy similar sensitivity to singleplexing with a greatly reduced workload.


Figure 2. HOT FIREPol® Multiplex qPCR Mix (Purple) was used to perform 4-plex or single-plex probe-based qPCR with five tenfold serial dilutions of human cDNA (cDNA concentration ranges from 200 ng to 20 pg per reaction). Reactions were performed with Applied BioSystems™ QuantStudio™ 6 cycler using Purple dye for normalization.