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I'm trying to use qPCR to quantify the amount of a specific bacteria species in patient sputum, but I don't really have the option to use consistent quantities of sputum to elute DNA from. The qPCR works fine, but once I have the Ct results, what is the best way to account for the fact that I'm using different masses of sputum?

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  • $\begingroup$ Are you always using a specific amount of DNA from your samples (i.e. 10 ng) or a relative amount (i.e. 10% of yield from one sample)? This make an important difference in how you can/have to normalise your results. $\endgroup$ – Nicolai Feb 13 at 9:54
  • $\begingroup$ Relative amount, like 10% of the yield from a sample. We expect human DNA and DNA from other bacteria to be in the sample, so diluting all samples to the same quantity of DNA may not be useful to us. $\endgroup$ – ZLB Feb 13 at 17:42
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If you are always using a relativ amount of your input yield for each sample, then you'll need to correct your Ct values for the absolute DNA yield of each sample. If you can't measure the DNA yield of each sample, then you'll just have to assume it is directly dependent on the sputum input mass. This assumption is pretty faulty though, since direct DNA measurements are much more exact and are not affected by biases from DNA extraction efficiency differences or batch effects.

Since Ct values represent a single qPCR cycle in the linear phase in which the target DNA is doubled, you need to adjust the CT values by +1 for samples with half DNA input and by -1 for samples with double input (or generally $Ct_{adjusted} = Ct - log_2(inputfactor)$ ). Of course this method of normalising assumes that the relative content of your target DNA in the sample is independent of the total sample yield (which may or may not be true; if your specific bacteria species is proliferating a lot more in one patient compared to the other this might affect both relative and absolute DNA amounts).

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