2-ΔΔCT interpretation of gene copy number - What does a 2-ΔΔCT value of 1.84 or 0.7 mean? (Apr/28/2009 )

Hello,

I'm trying to see if my gene has an increased copy number in tumor A and tumor B. I used DNA from normal tissue as a calibrator which gave a ΔΔCT value of 0 and therefore the 2-ΔΔCT is 1 which is great.

2-ΔΔCT (2 to the power of minus Delta Delta CT)

However, when I calculated the 2-ΔΔCT (which indicates the copy number) for my gene, in tumor A it gave a value of 1.84 and in tumor B, the 2-ΔΔCT was 0.7.

Does anyone know how to interpret these data? Am I missing something?

Shall I be mutiplying the 2-ΔΔCT value of my normal tisse (calibrator) by 2 since it has 2 alleles? and therefore multiply the rest of the 2-ΔΔCT vlaues by 2 also? or does the 2-ΔΔCT take that into consideration?

I guess my question is: what does 1.84 mean? that the gene has almost 2 copies only in tumor A? Isn't it normal to have 2 copies of the gene anyway? If so then what does 0.7 mean?

Any clue? I'm totally confused

Thanks alot

I've never done copy numer analysisi but I think you compare it usually to a single copy gene within the same sample.

Piano on Apr 29 2009, 12:32 AM said:

Hi

Did I get it right? You ask wether your ΔΔCT value gives you any idea about the copies of your gene in the genomic DNA? No, it doesn't. It is just about the number of mRNA copies you find in your sample, which could be high if the gene is more expressed than your control or low, if it is less expressed than your control.
The other explaination would be, that your control just doesn't work and freaks out. Then you could also measure differences, that do not exist.
Another possible explaination would be, that I just did not get the point of your question due to my limited brain capacities...

Cheers

Piano on Apr 29 2009, 12:32 AM said:

First ΔΔCT ct method uses target gene and reference gene. You only write about your gene of interest. Let's see if you have the numbers right.
The equation is:
relative normalised ratio= 2 power of <(Ct target - Ct reference) of calibrator - (Ct target - Ct reference) of sample>

Ratio for a calibrator alone when put into the equation has to be always one, that means 100 %. That means two copies, as the original copy number is 2.

If you did it this way, and your ratios for tumor A and B are 1.84 and 0.7, than it means 184 and 70 % of the calibrator copy number. That is 3,68 and 1,4 copies per cell.
Of yourse it doesn't mean that each cell has a three and a part of a gene, it has to be a natural number, but that some portion of cells have more than two copies.
In the other case (70 %) it means, that some of the cells have less then two copies in a cell.