FAQ: Can I run multiplex reactions with the Luna® Probe One-Step RT-qPCR Kits? Do I need to change my reaction conditions?

Yes, multiplex reactions amplifying up to 5 targets have been successfully run using the Luna Universal Probe RT-qPCR Kits. Select the different fluorophores corresponding to separate detection channels of the real-time instrument. Each target should be tested in a singleplex reaction first. For each amplicon, begin by including 400 nM of forward and reverse primer and 200 nM probe in each reaction. If performance of a particular amplicon is seen to deteriorate when used in a multiplex assay as compared to its singleplex results, reactions may require optimization. It can be helpful to reduce the primer concentrations for an abundant or high-copy target and/or increase the primer concentrations for the low abundance or low-copy targets. Further optimization may be needed as multiplexity increases, using the following parameters as guidelines: primer 100–900 nM, probe 100–500 nM. 

Example of a 3-plex reaction using the Luna Universal One-Step RT-qPCR Probe Kit on an Applied Biosystems™ 7500 Fast instrument: