transgene copy number determination with Q-PCR - (Oct/26/2008 )
Decided I should post here before I fry my brain completely on this matter . I did search the boards for this but didn't come across a clear answer.
I have stable transgenic cell lines that I want to characterise, to be specific, I need to determine the transgene copy numbers. I have to do this with Q-PCR, southern is not an option. I have the plasmids for these constructs, the maps and specific primers that would only amplify the transgenes.
My question is what is the best method to do this and can you direct me to a protocol/reference? (esp about the calculations).
- can i simply make external standards with linearised plasmids and estimate copy number in genomic DNA? if so how to calculate?
- or do i need an internal reference, say amplify a single copy gene for each sample and use its Ct to determine the copy number of the transgene? if so how do we do this and how do we incorporate amplification efficiencies etc into the equation.
all help appreciated!
thanks in advance
Need a internal reference gene to determine the DNA number. The transgenic cell have only one copy of allele (DNA) that shows higher delta Ct. The nomal ES cell have two copy DNA, hence having less delta Ct value.