Measurement of rAAV genome titre
Genome titre was quantified by digital droplet PCR (ddPCR). Aliquots of
post-lysis supernatant (5 µL) from total cell culture were treated with
DNAse I (Roche, Basel, Switzerland) to remove residual plasmid DNA.
DNAse I-treated samples were diluted 1000- or 10,000-fold in PCR
dilution buffer (GeneAmp™ PCR Buffer I (Thermo Scientific), 0.02%
UltraPure™ Salmon Sperm DNA Solution (Invitrogen, Waltham, MA), 0.1%
Pluronic™ F-68 non-ionic surfactant). Droplet formation and subsequent
post-PCR droplet analysis was performed using the QX200 system (Bio-Rad,
Hercules, CA), with absolute quantification of AAV genome copies/µl
determined using the Quantasoft analysis software (Bio-Rad). Genome
detection was achieved using primers and a FAM-labelled probe targeting
the PolyA sequence of the pAAV-CAG-GFP plasmid. Capsid titre
quantification was performed using the AAV8 titration ELISA (Progen,
Heidelberg, Germany) from total cell lysis supernatant diluted in 1x
ASSB assay buffer (Progen).