AAT Bioquest

What factors should I consider when I choose fluorophore/quencher combinations for my multiplex qPCR reactions?

Posted May 12, 2023


Designing an experimental setup for multiplex quantitative PCR (qPCR) is more complex than for single reactions for a number of reasons. The first is the need for probes that contain unique reporter dyes with distinct spectra to detect individual targets. Secondly, real-time detection systems vary in their excitation and emission filter settings across different manufacturers, requiring instruments to be calibrated for each dye as part of the experiment optimization process. Proper calibration enhances dye specificity and minimizes background noise and overlap of fluorescent signals.

Few things to keep in mind when selecting dyes for multiplex qPCR: 

  • Choose dyes that are compatible with the qPCR instrument you’re using - You want to make sure your qPCR instrument is capable of detecting the emission spectrum for each dye being used. 
  • Choose dyes that can be calibrated on the qPCR instrument you’re using - The real-time qPCR instrument must be calibrated for the set of dyes you choose. If it isn’t calibrated for your chosen dyes, you must calibrate it before you start your experiment or select different dyes. 
  • Avoid overlap of emission spectra - Choose dyes with appropriate excitation wavelengths and minimal to no overlap in their emission spectra. Take into consideration the total fluorescence intensity as well. If your instrument uses ROX (6-carboxyl-X-Rhodamine) as a passive reference dye, avoid using any dyes that have spectra overlapping with ROX. 
Additional resources

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore

Real-Time PCR (qPCR)

ROX Reference Dye *50X fluorescence reference solution for PCR reactions*