Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 35

Transfer the virus to a new 1.5 ml microcentrifuge tube.
Keep the appropriate number of aliquots for quantification and
quality control assays. To avoid repeated freeze-thaw, make
small volume aliquots and store the virus stock at −80 °C.

3.4 Vector Titration

(a) Quantitative PCR and Dot Blot Assay
The genome-containing particle titer (gcp) indicates the num-
ber of viral particles that have encapsidated the rAAV genome,
regardless of their infectivity or transgene expression. The gcp
titer can be determined by quantitative PCR or dot blot analyses
[ 60 - 62 ]. Titers are calculated from a standard curve generated
from the rAAV plasmid DNA. Recombinant AAV stocks are
first digested with DNase I to degrade any unpackaged repli-
cated vector genome and input plasmid DNA. The encapsid-
ated viral genome is subsequently released from the capsid by
proteinase K treatment.
(α) DNase I treatment
Viral DNA is prepared as follow for quantitative PCR and
dot blot assay.
Take a 4-μl aliquot of the purified rAAV virus.
Add 200 μl of DNase I buffer and 20 U of DNase I.
Incubate at 37 °C for 1 h.
Add 200 μl of 2× proteinase K buffer and 2 μl of proteinase
K. Incubate at 37 °C for 1 h.
Add 400 μl of phenol-chloroform (24:1) and vortex for
5 min. Centrifuge at 13,000 rpm for 5 min at 4 °C.
Carefully transfer the aqueous phase to a new 1.5 ml
microcentrifuge tube.
Add 1/10th volume of 3 M sodium acetate and 1 μl of
glycogen at 20 μg/μl to the sample, vortex. Add 2.5
volumes of 100 % ethanol, vortex. Precipitate over-
night at −20 °C. Centrifuge at 13,000 rpm for 20 min
at 4 °C. Carefully discard the supernatant.
Wash the pellet with 500 μl of 70 % ethanol. Centrifuge at
13,000 rpm for 5 min at 4 °C. Air-dry the pellet for
5 min.
Resuspend viral DNA in 40 μl of TE.
(β) Quantitative PCR.
Perform each viral DNA sample and controls in duplicate
to ensure accuracy.
Prepare a standard curve with linearized AAV plasmid
DNA. Dilute the digested DNA in Lactated Ringer's
Solution (LRS) at a concentration of 20 ng/μl. Make