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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!

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    $\begingroup$ 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, 2023 at 20:12

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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.

Edit (because I did not go all the way) : Then you can plot the Ct values against the log of the concentration of your standard. You extract the formula of this curve, and you replace x by the Ct value of your sample in order to know y = concentration of target in your sample.

Otherwise I found this here : sciencedirect.com/science/article/pii/S0301472X02008068 "Most real-time PCR instruments have software that can automatically compute the amount of template of an unknown sample from a standard curve. However, it can be done manually by putting the observed Ct value for an unknown sample into the formula: (observed Ct − y intercept)/slope." intercept and slope being the ones of the calibration curve.

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  • $\begingroup$ Thank you very much for the prompt, analytical, and clear answer. You reply without being ironic, trying to evaluate my skills, or asking me testing questions. I think, I understand the logic and the connection between the curve and the copy numbers. And regarding the first formula? Can this be used for direct copy number calculation? I am asking cause I want to understand how to connect the rest of the excel columns with copy numbers and ct? $\endgroup$
    – KGee
    Sep 25, 2023 at 20:00
  • $\begingroup$ I have never seen the first formula before. I do not know what type of samples you have run, what it is your columns contain. But what I know for sure is that you need to have a calibration curve in order to know the relationship between Ct and number of copies. $\endgroup$
    – CaroZ
    Sep 25, 2023 at 21:01
  • $\begingroup$ ... I just found here : sciencedirect.com/science/article/pii/S0301472X02008068 "Most real-time PCR instruments have software that can automatically compute the amount of template of an unknown sample from a standard curve. However, it can be done manually by putting the observed Ct value for an unknown sample into the formula: (observed Ct − y intercept)/slope." intercept and slope being the ones of the calibration curve. $\endgroup$
    – CaroZ
    Sep 25, 2023 at 23:45
  • $\begingroup$ Thank you once again for the reply as well as for the paper!!!!! Now I can practice myself on qPCR data!!! $\endgroup$
    – KGee
    Sep 26, 2023 at 19:08

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