I have been considering the number of MGB probes I can use in my multiplex real-time PCR. The conventional advice suggests using up to two MGB probes, with the third probe being 'normal' - without MGB. However, in my experiments, I tested three sets of gene-specific primers and MGB probes, and surprisingly, it worked perfectly. I did not observe any decrease in efficiency compared to a singleplex assay. I'm curious if others have experience with using three MGB probes and if they observed any reduction in reaction efficiency.
It depends on how any filters your instrument has and how well the deconvolution is programmed (the more filters the less different the detected wavelengths the larger possible crosstalk).
Do you really need the MGB moiety on your (TaqMan?) probe? They can help in some situations (like sequence variant discrimination) but if you are doing gene expression and you can design primers at the Ta/Tm that you desire without the MGB, in that case I think you can ditch the MGBs. MGBs boost the annealing temperature which can be desirable in certain situations. If you are using ThermoFisher for the MGB probes, consider other quenchers such as QSY.
As Jochen points out, bleed-through AKA crosstalk is a phenomena to consider here. If you make 3-4 plex, which channels do you expect to give emission? Just because you made a 5 plex doesn't mean you will get positivity in all channels all the time. If you manage the channels, you can manage the cross-talk (to a degree sometimes), but if you get positivity in all channels all the time then you will have some specificity issues/headache with analysis. Check your instrument manual on some combination recommendations, I know the RotorGene instrument manual gives some recommendation for channel combinations.
https://www.thermofisher.com/tr/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/what-is-multiplex-qpcr.html
Also check this tool that might help you visualize all these different channels:
https://www.biosearchtech.com/qpcr-multiplex-spectral-overlay-tool
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