Hello

It does not sounds right!

First you need to calculate the average of replicates belong to your inter-calibrator sample. Adjust any individual Ct of each run based on the difference you observed between runs.

It is not as simple as calculating the average Ct of all inter-plate calibrators.

For example in run#2, you have a +1 Ct in compare to run #1 for your calibrator, then to adjust run#2 based on run#1, you would consider subtracting 1 from Ct of all individuals in run #2.

Hope I was clear.

I believe it is very difficult to work with averaging between different experiments. It is more likely to screw up results if the variation between the experiments is high like in this example (2Ct, i.e. 4fold difference between the highest and lowest sample). Assuming that you had indeed the same amount of DNA in each experiment I would expect the whole rotor to be shifted by the difference of the reference sample. That means that you should look at the difference of Ct's of samples vs. reference DNA in the same rotor. So, the reference Ct should be 19 for rotor 1, 20 for rotor 2 etc. This will give you a relative difference for the other samples in this rotor.

Otherwise you will calculate an increase or decrease for different rotors even if the difference would be the same within each plate.

E.g. Sample X with Ct 20 in rotor one would mean 1 Ct difference, i.e. factor 2 if Ct 19 for reference DNA is used. If you use an average of 19.9, the difference is only 0.1Ct, therefore much lower and probably wrong, because you assume that sample X would have the same fixed Ct in another rotor (which it most likely doesn't).

I agree Ali Javadmanesh

Yes of course real tlme pcr

Thanks for your answers Ali Javadmanesh and Hüseyin Besir

I understood what both of you are suggesting. I will correct all of the samples in each rotor.

But my problem is that I have 5 rotors of the same experiment (many samples, same gene) and my inter-calibrator range around Ct= 1.2 between the higher and lowest (real data, not like my e.g.). Then, how I choose the right one to use to correct others?

Thanks for your help

As I wrote, each rotor is a separate experiment, with the same sample in all the rotors, each serving as reference for that particular rotor. The results from different rotors can be compared after calculating the relative amounts in each rotor.

Dear Sir,

How can I calculate CT of these values attached to this message.

Thanks