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Quantifying relative ratios of variants by qPCR

Hello,

I am quantifying relative amounts of different genomic variants in a population of cells. Basically, in a population, a certain percentage will be in state "A" and the remaining percentage in state "B". I have optimised qPCR primers to detect both states with equivalent amplification efficiencies.

For that, I extract genomic DNA from the whole populatio and then qPCR for state "A" and "B" separetly as well as another region that is stable (calibrator or reference for gDNA input). I can calculate the fold change in between these two states by deltadeltaCt.

However, I would like to express the fold change in relative percentage, so instead of reporting 0.4 of fold change of state "A" versus state "B", I would like to say that state "A" exist in X% in the population. Anyone has an idea how to do that? It should be similar to what folks working with siRNA have been doing, as is calculating % of knockdown of specific gene.

Thanks,

TP

qpcr deltadeltact
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updated by hazzardwallace • hazzardwallace • written by ThePresident ThePresident

And what happens if the difference is huge. For example, for one of my conditions, the state "A" is 36 folds bigger than state "B"... How could-I calculate percentage difference, i.e. able to report something like "population has 96% of state A"?

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written by ThePresident ThePresident

1 answer

Actually, it's pretty easy... By multiplying the Fold Change by 100 we get the relative percentage. So, fold change of 0.4 represents 40% of state "A" relative to state "B". I am just not sure how to deal with fold change > 1? It seems weird to report a percentage of 300%...

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written by ThePresident ThePresident

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