Quantifying Adeno-Associated Virus Titers

vg levels were quantified by qPCR, using primers and probes targeting ITR, cap8, capAnc82, or transgene sequences. 3 μL of AAV prep was incubated for 45 min, at 37°C, with 20 U of DNaseI (04716728001; Roche, Basel, Switzerland). Digested samples were then supplemented with 20 μL of 20 mg/mL proteinase K (740506; MACHEREY-NAGEL) and incubated at 70°C for 20 min. vgs were further extracted using the NucleoSpin RNA Virus kit (MACHEREY-NAGEL). qPCR was performed with a StepOnePlus Real-Time PCR (Life Technologies, Carlsbad, CA, USA). qPCR reactions were run in a final volume of 20 μL, containing the primers/probe PCR Master Mix (TaKaRa, Kusatsu, Japan) and 5 μL of template DNA. For ITR titration, the standard plasmid was prepared in accordance with D’Costa et al.²¹ (link)