I have seen some papers, indicating they they have used different primer/probe concentrations in their multiplex qPCR experiment. For example, 900nM forward primer, 300nM reverse primer, 100mM probe for one set, and 900nM forward primer, 300nM reverse primer, 300nM probe for another. I have attached a paper as an example.
I understand that you need to optimise a multiplex assay but is this 'cheating'? As in, they are biasing a target, since one target has more resources to get amplify (vice versa). Or have I misinterpret this?
Different primers will have different efficiency of amplification (you can observe this for yourself by setting up a serially diluted standard curve). For primers that poorly amplify, extra primer is needed to bring their efficiency as close to 100% as possible.
This is not cheating, rather this is a way to normalize bias in these experiments because if you had several multiplex primer sets amplifying their targets at different rates, your results would be skewed and, consequently, difficult or impossible to interpret.
I completely agree with Govinda. You want your amplification products, regardless of how many different products they are, to amplify within your 30-40 cycles. If, everything else being equal, you have one product that amplifies at a much lower efficiency than the other, and the first already amplifies poorly, you might never see the former show up.
Ideally, once you have an optimized protocol (probe/primer concentration, temperature cycling) for both reactions simultaneously, you make sure you use that protocol across all your samples/experiments. In theory, one of the signals of the multiplex should be the normalizing metric across them all (if that's your aim).
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