Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 27)

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2. If the lower expressing assay has a longer amplicon, >150 bp, then consider redesigning the
assay to be shorter or equivalent in length to the higher expressing gene. Shorter amplicons
typically can have greater PCR efficiencies.
3. Choose assays with more similar expression levels, if possible, to avoid reagent
competition. If this strategy is not possible, optimize the assays using a primer-limiting
strategy to limit the available primer for the higher expressing gene. This in turn forces an
earlier plateau phase of PCR.
a. Construct a primer matrix (Table 6.) for the higher expressing assay ranging from 50 nM
to 150 nM while keeping the lower expressing assay constant.
b. Select the concentration that generates the lowest fluorescence signal without any effect
on the Cq compared to singleplex data.
c. Repeat the multiplex experiment to compare the newly optimized primer set.
Table 6. Primer matrix.
Forward Primer, nM
Reverse Primer, nM 50 100 150
50 50/50 100/50 150/50
100 50/100 100/100 150/100
150 50/150 100/150 150/150