I recently started to get involved with quantitative PCR. I have some data and I want to calculate the copy number of them. In the output Excel file, I have the following cells (I am running Quandstudio v.1.5.2).

Well| Well Position|Omit|Sample Name|Target Name|Task Reporter|Quencher|CT|Ct Mean|Ct SD|Quantity|Quantity|Mean|Quantity|SD|Y-Intercept|R(superscript 2)|Slope|Efficiency

I found the following formulas

copy number = 10^(ct - intercept)/(slope) 
copy number = 2^-ΔΔCt

I have two questions. a) Are these formulas accurate? b) Is there any other suggested formula that I can apply to my data?

If there is any helpful article that has calculation formulas, it is more than welcome!

  • 1
    $\begingroup$ Welcome to the Biology Stack, this site values questions that show some attempt to research the answer for them selves. So - what can you tell us about the two formulae - What situations would you apply them in? Why are they different? $\endgroup$
    – bob1
    Sep 14 at 20:12

1 Answer 1


The second formula (2^-ΔΔCt) you are presenting does not calculate a number of copies of your target. It is instead used in relative quantitation, where you want to know the relative expression of your gene of interest normalized to a reference gene, and compare it between samples of different treatments.

From your question, it seems that you want to perform absolute quantitation instead, more precisely, you want to know about the exact number of copies of your target in different samples. For this, you need to run your samples alongside a standard curve consisting of several dilutions of a known quantity of your target. You have to know the number of copies of the target which are present in each dilution step by following the method described here: https://toptipbio.com/dna-copy-number-qpcr/

After running your plate and provided the efficiency of your primers is ~ 100 % and is similar between your standard curve and samples (which you should have tested before), you now know the relationship between number of copies of the target and Ct value. The Ct value you have in each sample can thus be extrapolated to a number of copies.

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