Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 10)

4 | SsoAdvanced ™ Universal Probes Supermix Instruction Manual 4 |
Multiplex Assays Design Considerations
To ensure data generated in a multiplex reaction are equivalent to data generated in a singleplex
reaction, it is imperative to evaluate the assay performance in both singleplex and multiplex
reactions. It is also important to understand the expression level of your target sequences, as
this will impact the multiplex optimization method.
1. Determine the expression levels of the genes prior to optimizing a multiplex approach. This
can be accomplished through the use of standard curves derived from template serial
dilutions (for example 100 ng to 1 pg).
2. Assign the reporter dyes based on the expression levels; brighter fluorophores should be
reserved for lower expressing targets.
3. Consider these factors when designing primers and probes:
■ ■
■ ■
Analyze all oligos for primer-dimer stability with all other oligos in the reaction
■ ■
Account for nonspecific primer-probe annealing and cross reaction between assays
■ ■
Amplicons of 50–150 bases are preferred. Shorter amplicons often have better
PCR efficiencies
4. Optimize each primer set.
■ ■
Not all primer sets/concentrations perform the same. Empirical testing using a standard
curve is imperative
■ ■
Prepare two standard curves for each assay
– One singleplex for each assay (if not completed already)
– One duplex (or multiplex if applicable)
■ ■
PCR efficiencies must be similar to minimize amplification bias
■ ■
When combined in a reaction, be sure each assay is used within the appropriate template/
target dynamic range. This range is often reduced when used in a multiplex application
Fig. 1. Graphs show the three modes for expression in a duplex reaction. A, both genes express relatively equally;
B, one gene always expresses more than the other; C, one gene varies in expression levels depending on the sample.
A B C
All assays in a multiplex reaction should have the same or nearly the same annealing temperature