I run qPCR to titer the AAV ( which i got by transfection in 100mm dish). I make four dilution of my sample ( 12 well and triplicate). I take GFP plasmid as a standard and were diluted to 1ng/ul, accordingly, 8 dilution to 0.05ng/ul (24 well and triplicate). Now i got the Ct, Ct Mean, Ct SD, and quantity. I need to calculate the the quantity in picogram/well, picogram/ml, genome copy and genome copy per ml. kindly pls suggest me any way, how to calculate it. Thanks.
Hi,
Assuming you were detecting a component specific to the vector, try plotting Ct mean of your standard curve (y axis) vs. Log(pg plasmid) and get a linear equation for best fit. Intrapolate log(pg) of your titering samples, then anti-log to get a pg value. Adjust for the dilutions and/or volumes accordingly.
To determine a per genome value, you presumably have Ct mean values for a genomic marker? You can use an average of those to normalize your standard curve values, and then to normalize your titering samples.
To orient you, Ct values and vector copy number have an inverse relationship.
Hope this helps
Hi,
as stated above, you should calculate the copy number of your standard plasmid. For our standard plasmid 1 ng/µl would correspond to ~2x10^8 copies (note, that you don't need a correction factor of 2 in this case because you count both strands of the plasmid, so the same as counting the ssDNA genome of the AAV).
This should help to setup your dilution series (e.g. 10fold over a range of 10^8-10^3). Your range for the standard seems too narrow to me, if your final dilution is really 0.05ng/µl, that's only a factor of 20 from your starting conc. Remember that qPCR has a wide dynamic range and it would be very difficult to hit a range of 20 with your dilution of an unknown sample.
I guess you have a software for the qPCR machine so you should be able to calculate the unknown sample and multiply with the dilution factor to get the initial concentration. Once you define your standard series and their concentrations, the software calculates all unknown samples by itself. Reading the manual can help in this case.
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