I would like to compare the data between Taqman Real time PCR and Taqman Digital PCR using 6 samples and 8 target genes. I want to know whether delta delta Ct is ok or not for comparison. I also want to know which method is the best for calculation of delta delta Ct.
If this is your question than don't use some target genes which relative expression is unknown. Create mock samples with well-defined and known relative expressions. Then you can study and compare not only the precision but also the accuracy of both methods. Otherwise your conclusions will be of verry little relevance.
The question for "the best method" to calculate delta delta Ct needs some specification. The "method" here is a simple subtraction. There are no other "methods" and the question of "the best one" is not really sensible. Could it be that you mean the entire process of calculating the Ct values (background subtraction and detranding of signal curves, smooting, algorithm to determine the Ct, consideration of amplification efficiencies, ...? The literature is full of papers about that and most of them suffer the same problem that "something unknown" is measured, so assertions about accuracy can't actually be made.
If this is your question than don't use some target genes which relative expression is unknown. Create mock samples with well-defined and known relative expressions. Then you can study and compare not only the precision but also the accuracy of both methods. Otherwise your conclusions will be of verry little relevance.
The question for "the best method" to calculate delta delta Ct needs some specification. The "method" here is a simple subtraction. There are no other "methods" and the question of "the best one" is not really sensible. Could it be that you mean the entire process of calculating the Ct values (background subtraction and detranding of signal curves, smooting, algorithm to determine the Ct, consideration of amplification efficiencies, ...? The literature is full of papers about that and most of them suffer the same problem that "something unknown" is measured, so assertions about accuracy can't actually be made.
Hello Jochen Wilhelm, thank you very much for your answer. But our main purpose is to detect gene expression of the Target genes using 6 samples. We usually use the Real time PCR for it but we recently bought a new digital PCR machine and we want to compare the data between these two methods. Thank you very much for your excellent answer.
It might be that you wish to compare the methods for your genes... but: you won't learn anything more than which method will have the better precision. Even if both methods give the same result, this result can be equally wrong for both methods. Or both results may be different, then you won't know which one is better. The accuracy can only be analysed when you know the "true" value.
ic, thank you very much. Can you tell me how to create the mock samples with well-defined and known relative expressions? If you have any examples of the test, please kindly teach me.
If you analyze human samples you may use for instance bacterial plasmids as standards. Make a miniprep, open the plasmids, purify and quantitate them, for instance by OD260 or by a fluorimetric method. Alternatively, you can make a PCR to gerenrate PCR product (of a bacterial, fungal or plant sequence). I recommend to generate a standard that is longer than the product to be amplified in the qPCR. Dilute the purified standard to a desired concentration and spike a defined amount to your human samples. In order to do a relative quantification, you will need two standards (two different sequences), one serving as "gene of interest", the other serving as "reference gene". This whole procedure is actually straight-foreward and simple. It is just to simulate the presence of some sequences in your samples from which you do know the exact quantity (because you generated, purified, quantified and spiked them).
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