The AAV genome titer [viral genomes per mL, (vg/mL)], also called physical titer is typically measured via quantitative real-time PCR (qPCR), the golden standard method to quantify the concentrations of cDNA or gDNA of a sample2. The relatively fast, easy and cheap technique provides information about the number of capsids containing the transgene of interest with a high through-put. However, several issues including sample processing, dilutions of the standard material, primer design, primer binding, or PCR efficiency can cause high inter-laboratory or inter-assay variations of the results for identical samples1.
Digital-droplet PCR (ddPCR), a more recently emerged DNA detection technique, deals with some of the limitations of qPCR. The principle of this method is the partitioning of the sample into thousands of droplets to separate single DNA molecules and the individual PCR amplification in each subdivision. Since the measurement is performed in one preparation, pipetting errors and contamination problems are minimized. This technique enables a highly sensitive absolute quantification of concentrations without dilution series of standard DNA with known concentrations, as necessary for qPCR5. Unfortunately, beside the high costs for the equipment, variations between labs can still occur due to different sample processing protocols.
