gravatar for ebs15242

6 hours ago by

I'm trying to understand how to present variation qPCR data when using the ddCT method.

I'm reading ABI's Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR that uses an example pulled directly from Livak 2001.

I follow how they propagate error through the Ct1-Ct2=dCt calculation, by squaring the standard deviations, summing them, and then taking the square root. That makes sense.

s = (s1^2 + s2^2)^(1/2)

But when they do the second subtration, they don't use the same formula. They say:

The calculation of ddCt involves subtraction of the dCt calibrator
value. This is subtraction of an arbitrary constant, so the standard
deviation of the ddCt value is the same as the standard deviation of
the dCt value.

This makes no sense to me. How is the calibrator value an "arbitrary constant", when it's measured and has uncertainty? In their example, it has even more uncertainty than the experimental value.

Is this approach correct? Why do they not use the square root of the sum of the variances like they did the first time?

Source link