Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 11)

| 5 SsoAdvanced ™ Universal Probes Supermix Instruction Manual | 5
5. Reduce the primer concentration of the higher expressing target.
■ ■
Reducing the concentration, often times to 100–150 nM final, enables the lower expressing
target to amplify sufficiently. Use a primer matrix to determine the optimal concentration,
at which the chosen concentration would yield no shift in Cq values while exhibiting the
lowest flwescence signal
6. Reduce the primer concentration of both or all targets when the expression levels are
unknown or vary from sample to sample.
Notes: Preferential amplification of targets:
■ ■
Target abundance should not vary too much between assays; limiting variance guards
against domination of the reaction by a more abundant target
■ ■
Verify that the Cq values have not changed between the singleplex and multiplex
standard curves
– If unavoidable, it may be permissible if all assays in the reaction shift equally
Fig. 2. Overlay of singleplex and duplex standard curves demonstrating no
significant performance differences, thus a well-optimized duplex exists.
40
Cq
35
30
25
20
15
Log Starting Quantity
Duplex Singleplex