Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 108

9. Spin 20,000 × g for 5 min. DNA is in the upper layer. Transfer
450 µl of the upper layer to a new tube.
10. Add 450 µl 100 % chloroform and vortex.
11. Spin 15,000 rpm for 5 min. Transfer 400 µl of the upper layer
to a new tube. Theoretically 80 % of total viral DNA will be
recovered.
12. To the 400 µl DNA (p/c extracted supernatant), add 40 µl
3 M sodium acetate (pH 5-6), and add 1,000 µl of 100 % etha-
nol and 1 µl glycogen (20 mg/µl). Incubate at −80 °C for
30 min. This step is to precipitate the DNA.
13. Spin at 15,000 rpm for 40 min at 4 °C. Discard supernatant.
There should be a white DNA pellet in the bottom.
14. Wash the pellet with 500 µl 75 % ethanol and spin 15,000 rpm
at 4 °C for 5 min.
15. Discard the supernatant and dry pellet for 5 min.
16. Resuspend the pellet in 50 µl sterilized H 2 O. Use the DNA as
a template for real-time quantitative PCR (RT-qPCR).

Standard RT-qPCR can be applied for calculating the number of
viral particles. We amplify the WPRE region using a set of primers
as follows:

Real-Time qPCR
for Quantifi cation of rAAV
Genome Copy Number

WPRE forward, 5 ′ -ACTGTGTTTGCTGACGCAAC-3 ′

WPRE reverse, 5 ′ -CAACACCACGGAATTGTCAG-3 ′

Annealing temperature: 52 °C

We defi ne the number of molecules per 1,000 bp/1 µg as
equal to 9.1 × 10 11 .

pAAV2-MCS-WPRE = 5,829 bp, 1 µg of pAAV2-MCS-WPRE =
1.56 × 10 11

One copy of pAAV2-MCS-WPRE (double-stranded plasmid
DNA) is equivalent to two single-stranded AAV viral genomes.

1 µg of pAAV2-MCS-WPRE = 3.12 × 10 11 of AAV viral genomes
Therefore, 320.5 ng of pAAV2-MCS-WPRE = 10 11 of AAV viral
genome copies

The suggested range for viral particle (vp) load for injections is
1 × 10 10 -10 12 vp/ml, although this should be optimized according
to your protocol ( see Note 6 ). For example, if you are to inject
1 × 10 11 vp/ml, and your virus concentration is 128 ng/ml by
PCR, it would be 0.4 × 10 11 vp/ml. Thus, the viral prep has to be
concentrated 2.5-fold. We use Amicon Centriprep with
Ultracel-100 membrane for viral stock concentration.

Calculation of Physical
Titer (Viral Particles/ml)