Hi,

1. each pair of primers should be specific for your target region.

2. the ampliconn sizes should be different - to recognize what have you detected.

3. the primers should not be complementary to each other.

It is important to have the concentration of primers for the shortest amplicon lower (less volume) than concentration of primers for the longest one (more volume). It is because the shorter targets are amplified faster (preferably).

Good luck.

Hi Fahimeh Mohammadi,

I agree completely with Marcela Krutova, as the mentioned points, 

and also i think that the annealing temp. is the most important parameter for this experiment , this temperature must be suitable for all targets amplification.

Please you can read this reference

  http://www.premierbiosoft.com/tech_notes/multiplex-pcr.html

Good luck.

Hi!  If you are planning on multiplexing (multiple targets in the same well), then you will need to use the Taqman probes / probes, so that you can tell the difference in the signal from different primers.