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I am reading my qPCR results and having no idea why Ct value is returned like this:

enter image description here

This is the qPCR curve of my samples, and the software returned Ct values of most of the curves as seen: 33.99-38.4 for the curves that have lower baseline. The problem is the 2 curves (navy and black) with higher baseline at the first place. I read Ct values of these curves as 32-33, but the software says that the Ct values are 35.4 for these curves.

What could be the problem and what would be the right answer, my values or the software's? Thanks in advance!

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The software is correct. $C_t$ values are chosen to be sufficiently above background. As you can see, the background level is very different for two of the curves, so you can't use the same signal threshold for determining their $C_t$.

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  • $\begingroup$ Thanks! I understood that the threshold for the black and navy curves should move up as the baseline is higher. $\endgroup$ – Kidan Aug 21 '17 at 9:58

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