Primer-dimer is one of the things that makes multiplexing so challenging.

My advice is to take a close look at the 3' ends of all the primers and make sure that none of them are complementary to any of the others. Remember that G:T is also a stable basepair.

Hot start helps, but the high primer concentration will partially negate this and once a single dimer forms it will amplify very efficiently.

I try to make sure that my primers all end in AC. If you can achieve this, you are well on your way to preventing primer-dimer, though there is still the possibility of an internal match.

But if you need maximal sensitivity for all targets and these are not present in equimolar amounts, the best solution is simplex PCR. Targets with high concentrations outcompete low-concentration targets because they use up the reagents before the low-concentration target reaches a detectable level.

the simplest way to minimise p-d is to use a hot start enzyme so the primers never get to a low temperature while in the presence of an active enzyme. Using less primer will help also

How do you know that you have primer dimers? In a gel? Before test the assay, you use a primer online analysis tool to avoid major complications, like this one:

https://eu.idtdna.com/calc/analyzer

here you can check mathematically your probability pimer dimer like Hairpins, self dimer etc.

Before performing a multiplex test, test the singleplex reaction by different annealing temperature ( if your PCR device can do a gradient it is much more easier) (always do NTC and positive controls ).

Another step could be a primer concentration matrix to test the primers asympotically.

try tool for primer-dimer detection:

http://primerdigital.com/tools/PrimerList.html

Primer-dimer is one of the things that makes multiplexing so challenging.

My advice is to take a close look at the 3' ends of all the primers and make sure that none of them are complementary to any of the others. Remember that G:T is also a stable basepair.

Hot start helps, but the high primer concentration will partially negate this and once a single dimer forms it will amplify very efficiently.

I try to make sure that my primers all end in AC. If you can achieve this, you are well on your way to preventing primer-dimer, though there is still the possibility of an internal match.

But if you need maximal sensitivity for all targets and these are not present in equimolar amounts, the best solution is simplex PCR. Targets with high concentrations outcompete low-concentration targets because they use up the reagents before the low-concentration target reaches a detectable level.

The answer may come a bit too late, but I had success by reducing primer amounts and at the same time increasing elongation time and reducing elongation temperature (from 72°C to 68°C). The following paper helped:Article Novel approaches to mitigate primer interaction and eliminat...

I also used Multiplex Manager (http://www.multiplexmanager.com/) to analyse possible primer combinations and check for primer interactions.