I think I misunderstand the fundamentals regarding quantifying DNA copy number: When quantifying DNA copy numbers using droplet digital PCR (or qPCR) why don't researchers normalize to total DNA in the sample (using Qbit for instance) instead of of a reference (or housekeeping) gene? Thanks in advance!
Hi,
the principle applied for quantification of ddPCR data is to divide the amount of the target gene by the amount of the reference gene. NB: log(A/B) = log(A)-log(B).
Hope this may help you.
Yes, that is how you should do it.
If you need an absolute number of molecules, the only way to do that is to use an external standard, such as a purified plasmid. The number of molecules in the absolute standard is quantified exactly as you say. Once you know the number of molecules in the standard, you can use it in qPCR to calculate the copy number in impure mixtures, or any situation where you can't have a reliable Qbit reading.
Reference genes are used for relative quantification. That's when you want to make a statement like "my gene of interest is upregulated x-fold after drug treatment". You could try to normalize that too to the absolute input cDNA concentration measured by Qbit, but that would leave you with more unexplained variance than normalizing to a reference gene does.
The response by John Schloendorn is absolutely correct and explanatory for your query. I would just like to add one more point:
You can amplify the gene (from same genomic DNA sample) for which you are sure about the copy number instead of purified plasmid. It also works well.
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