Hello every one,
It is a little bit confusing for me to understand the numbers resulted from 2 to the power (-∆∆CT) method.
If I performed this method like this (for copy number variation study):
sample 1 (is case 1) > 2 to the power (zero) = 1.
sample 2 (case 2) > 2 to the power (-4.11) = 17.3.
sample 3 (case 3) > 2 to the power (4.9) = 0.03.
My question is:
for case 1 does the number (1) means this sample has the same number of copies like in it's match control?
for case 2 does the number (17.3) means it has copies more 17 times than it's match control?
for case 3 does the number (0.03) means this sample has less copies than it's match control? if less, by how much it less?!
Another question:
If these results are for gene expression, what does they mean?
Any help!!
Thanks in advance.
Maram
Hi,
The ∆∆CT method only works preferably for relative gene expression quantification and the 2 base is referred as the PCR efficiency, which means that during PCR one template generate exactly two fragments, which is not always the case . If you want to perform copy number experiment you should make a titration curve with the qPCR target (full-length DNA template amplified during PCR) at different very well known concentrations and then by linear regression infer the copy number of target sequences in your sample from titration Ct values. IN these experiments you will be able to obtain a proper efficiency parameter (2 or probably lesser)
The number you obtained mean the fold-change indicating how many times is your target over- or down-expressed when compared against your reference sample.
Good luck
Hi Alfonso,
Thanks for your response. It is clear for me now for the gene expression part.
But for the copy number we don't want to measure the (exact) copy number in the samples we just want to estimate if the cases can contain more or less copies than their matched controls, Is it not possible either?
Hi again Maram,
Copy number experiments require extremely accurate approaches to validate your hypothesis. That's the reason I recommend you to make absolute quantification of your target DNA and not the relative one. Additionally, you must precisely control the amount of DNA in your sample in order to properly normalize your Ct values. The eventuality you propose must be assessed in this way, otherwise you could have no correct estimations for your experiment, no matter if this is an exploratory study.
Alfonso
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