Absolute quantification of AAV genomes from total DNA with digital droplet PCR (ddPCR)

Accurate and absolute determination of AAV transduction from total DNA is important for understanding gene transfer efficiency across multiple experimental manipulations. As compared to relative measurements (e.g. through comparison of qPCR CT values between experimental conditions), absolute quantification can provide biological insights (e.g. actual average MOIs) that can guide further development of these vectors. This protocol enables absolute measurement of AAV genome number from total DNA samples extracted from animal tissues. Because total DNA is extracted, it is possible to estimate the number of AAV genomes per cell, using DNA content per cell and an estimate of transduction efficiency.