I suppose you can include all the reference samples in same run, but you need to use single set of primers for specific run. It would be very hard and technically not good to compare qPCR data for different set of primers.

Can you please elaborate what do you mean by this - If it is, how can I define reference samples for their relevant targets before run?

Hi Abhijeet,

Thanks for your reply. I have 3 different DNA from cell lines (MDA, Sol8 and NIH3T3) as triplicates (independent experimental repeats)). I have two reactions for each, one (A) is with nuclear primers (reference), the other (B) is with mitocondrial primers (target) and ultimate aim is to calculate B's copy number according to A's which is one copy. What I am asking is if I can run all the samples in one run? See the image for instance. You can see reference and target samples next to each other. Each also has two repeats. I hope that I make it clear.

If you say this is not possible or not accesible, how do you advise me to design the plate?

Thanks in advance

The plate design looks good. I would suggest you to include the positive control also, which means any marker which you know the copy number is same for nuclear and mitochondrial gene. If not possible, make triplicates for each sample. That could give you stronger statistical evaluation. Since you have enough space in your plate, I suppose you can do it.