I strongly recommend doing a training, visit a course, read a book (or some books) and work through tutorials to learn that technique. There are so many possible pifalls that you are likely to mess everything up when you don't thoroughly understand the whole technique - and RG is not the place to learn this. If you do learn and you get stuck with a very specific problem, you are welcome to come back and ask.

Please check this link, it may be helpful:

https://bitesizebio.com/24894/4-easy-steps-to-analyze-your-qpcr-data-using-double-delta-ct-analysis/

Hi,

I agree that qPCR is a complex topic which requires training, experience and optimized protocols as well as precise handling. In an attempt to answer your question:

The absolute amount of material that you will obtain through PCR for each sample for each primer pair is inversely proportional to 2^CT.  Therefore, you normalize the genes you are interested in to one (or better more) reference gene(s) within each sample to ensure that you don't run into any systematic errors due to differences between each sample.

Most frequently, the deltadeltaCt (ddCt) method is used with:

dCt=Ct(target gene)-Ct(ref gene) and

ddCt=dCt(target sample)-dCt(ref sample)

You calculate the ratio between your two samples by calculation of the quotient of target and ref.

Ratio=A/B

A: 2^(CT(target, untreated)-CT(target, untreated))

B: 2^(CT(ref, untreated)-CT(ref, treated)

Be careful in the choice of your housekeeping (reference) genes and throughly test your primer pairs for all reactions using a standard curve obtained from a serial dilution of your template. If your amplification efficiency is not 100% you should consider this in your calculations because for each PCR reaction you might only generate lets say 1.8 copies of your template rather than the theoretical 2 copies in the ideal case. Your new ratio would be then:

Ratio=A/B

A: Efficiency(target)^(CT(target, untreated)-CT(target, untreated))

B: Efficiency(ref)^(CT(ref, untreated)-CT(ref, treated)

In case you use multiple ref genes you can try the BestKeeper excel tool developed by Pfaffl et al. which uses basically the geometric mean of the ref genes.

thank you Kim Fabiano Marquart, Ahmed Gad and Jochen Wilhelm

https://www.researchgate.net/post/How_can_I_Calculate_expression_fold_with_CT_values