Confused about normalization - (Mar/15/2013 )

My girlfriend asked me to help analyze her qPCR results, but I have a math background and not a biological sciences one.

I'm a bit confused about how to normalize her data. I've include a subset below. (I know the efficiency values are weird/impossible, but someone else in her lab ran the standard curves)

Efficiency Values --> Target = 2.088, Reference = 1.99


CT(target) CT(ref) Concentration (target) Pfaffl Ratio Normalized Concentration

24.84 23.85 47600 2.055 ?
25.5 23.85 32900 1.264 ?
23.22 21.07 124000 * 1 124000
23.27 20.94 120000 0.881 ?
26.67 21.68 17500 0.120 ?


Using the Pfaffl method, I chose the sample with the largest concentration(*) to use as the "Calibrator"
I then applied the Pfaffl method to each sample to get the ratios above.

Now my question is how do I get the normalized concentration values? I thought that you multiply the concentration of the calibrator by the ratio calculated in each row,
but it seems strange that the data is so much different than the measured values in the first 2 samples when I do that.

You don't get concentration. Relative quatification (delta-delta method, Pfaffl method,...) only gives you a ratio. That's all that you can get from the equation.

If you want concentrations, you need to have a standard curves in-run, with known concentration values and calculate it by a standard-curve method (or usually directly by software).

The fact that the software spits out some concentration values is not important, there is no way the software knows the exact concentration in the first place, so there are some dummy values, you can't normalize them.

Efficiency is maybe theoretically impossible, but relatively common to get, especially with small amplicons

https://docs.google.com/file/d/0B15muZJPLjJgZ2VhV3pNRnFweUk/edit?usp=sharing

https://docs.google.com/file/d/0B15muZJPLjJgcGJwZzd1cC13MTg/edit?usp=sharing

https://docs.google.com/file/d/0B15muZJPLjJgeTQxSVgxNExOR0U/edit?usp=sharing