Hi Goran, it will depend on how you run the qPCR and on your machine.

If you are planning to use SyberGreen, you can only use one pair of primers because there is no way to distinguish between different amplicons, or, better, you will be able to distinguish them at the end of the reaction doing a melting curve if they are sufficiently different in length and/or base composition, but it will not be possible to quantify them separately during the run.

If you will design specific probes (TaqMan, Scorpions and similar) then you can run multiplex qPCR if your cycler allows it, meaning you need to have multiple excitation sources (typically led lights of different wavelengths) and a detector able to measure them. We typically run duplex reactions with FAM labelled probes (green, 496) and a HEX labelled reference gene (red, 538), because we don't use the ROX calibration that some cyclers require, but there are more dyes shifted to the red (Cy3, 550; Tex, 615; etc). Have a look at the link below, for example...

One recommendation is that if you want to multiplex, you run the calibration curve for each probe singularly AND in multiplex and you verify that it does not change significantly, otherwise it means there is some interference between the probes and this alters or removed reliability of your quantification (because the interference might change depending on the amount of each one of the templates you have in the sample).

http://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2014/01/14/recommended-dye-combinations-for-multiplex-qpcr

Hello

It is better you determine this number through benefit from the articles have a similar experiment for your idea . may help you these links

Also, I want to confirm you  about the nucleotides length from any  Primers In general, primers should be 18–24 nucleotides in length this provides for practical annealing temperatures .

Good Luck

http://www.bio-rad.com/en-us/applications-technologies/qpcr-assay-design-optimization

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Hi Goran, it will depend on how you run the qPCR and on your machine.

If you are planning to use SyberGreen, you can only use one pair of primers because there is no way to distinguish between different amplicons, or, better, you will be able to distinguish them at the end of the reaction doing a melting curve if they are sufficiently different in length and/or base composition, but it will not be possible to quantify them separately during the run.

If you will design specific probes (TaqMan, Scorpions and similar) then you can run multiplex qPCR if your cycler allows it, meaning you need to have multiple excitation sources (typically led lights of different wavelengths) and a detector able to measure them. We typically run duplex reactions with FAM labelled probes (green, 496) and a HEX labelled reference gene (red, 538), because we don't use the ROX calibration that some cyclers require, but there are more dyes shifted to the red (Cy3, 550; Tex, 615; etc). Have a look at the link below, for example...

One recommendation is that if you want to multiplex, you run the calibration curve for each probe singularly AND in multiplex and you verify that it does not change significantly, otherwise it means there is some interference between the probes and this alters or removed reliability of your quantification (because the interference might change depending on the amount of each one of the templates you have in the sample).

http://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2014/01/14/recommended-dye-combinations-for-multiplex-qpcr

In one real time run you can make multiplexing using different sets of primers and probe , it hard to optimize but with one important condition that all probes  have different excitation and emission wave length or different color in order to detect them together.