Quantifying relative ratios of variants by qPCR
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5.0 years ago
ThePresident ▴ 180

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 • 1.9k views
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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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5.0 years ago
ThePresident ▴ 180

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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