The most important aspect of multiplex qPCR to consider is potential negative interactions between PCR components, specifically the primers. Primer dimer formation can significantly decrease amplification efficiency. Therefore, you should analyze heterodimer potential between all of the primers included in your reaction to ensure that any structures predicted to form will not be strong enough to significantly affect the reaction. As with primer design for a singleplex reaction, strive for a ΔG value more positive than -9 kcal/mole for all predicted heterodimers. The IDT SciTools® OligoAnalyzer®
application is ideal for this purpose.