I have LCMV RNA samples of unknown concentration and I want to know how many LCMV RNA copies/mL I have in each sample. To do so, I am going to use the same volume of RNA/sample to perform a One step RTqPCR (Qiagen) and I will use a LCMV cDNA standard curve.
Such standard curve will give me the cDNA copies/mL in my samples. How can I then extrapolate the RNA copies/mL? Otherwise is it accepted to represent LCMV virus quantification as cDNA copies rather then RNA copies?
In any case you quantify cDNA copies.
If you want to know how many RNA copies correspond to one cDNA copy, you need to somehow determine the RT-efficiency. You might achiev this using synthetic RNA of which you know the exact concentration. But this is possibly like taking a sledgehammer to crack a nut. It might be sufficient to assume something like 80-90% RT efficiency.
You use the slope of the standard curve to determine the number of copies in each of your samples.
FYI, you cannot extrapolate (use data points that are outside the linear range of your standard curve). You can only interpolate (use data points that fall on the linear range of your curve).
Good luck!
Hi Vale,
When I am quantifying RNA copies/mL (e.x. HIV vRNA), I use plasmids with the targeted sequences (e.x. pNL4-3) as standards.
If you want the details, please let me know.
Thanx,
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