Biology Reference
In-Depth Information
3. hmNPCs are differentiated by passing them in dopaminergic
medium when they are 100 % confl uent.
1. HD adenoviruses are produced as in Chapter
15
.
10
11
-
10
12
CsCl purifi ed optical viral particles are incubated with 5 U
RNase-free DNase I in DNase reaction buffer, 1 h at 37 °C, in
order to remove contaminating DNA. DNase is then inacti-
vated by 30 min incubation at 75 °C.
2. Viral capsids are disrupted by incubation with 100
3.2 Extraction
of Adenoviral Genomic
DNA from CsCl
Purifi ed Viral
Preparations
μ
g protein-
ase in proteinase buffer in a fi nal volume of 200
L, 1 h at
37 °C. Proteinase is inactivated by 20 min treatment at 95 °C.
μ
1. A standard curve is generated from
Pme
I-cut pC4HSU or
pC4HSUgfp plasmids. Serial dilutions of the plasmids are pre-
pared ranging from 10
2
to 10
7
copies. Plasmid copies are cal-
culated considering the plasmid size (28,060 bp pC4HSU and
29,859 bp pC4HSUgfp), the equivalency between bp and Da
(1 bp = 635 Da), and the number of molecules per mole
(Avogadro's number, 6.022 × 10
23
). For pC4HSU, the molec-
ular weight is calculated as 28,060 × 635 Da/bp = 17,818,100,
therefore the molecules are (
3.3 Quantifi cation
of HD Adenoviral
Vector Genomes
g/17,818,100) × 6.022 × 10
23
.
The reference curve is obtained by linear regression of the
quantifi cation Cq values (
y-
axis) versus the log of molecules
number evaluated in each dilution (
x-
axis). PCR effi ciency is
calculated as
E
= 10
(−1/slope)
−1. Optimal amplifi cation effi cacy is
between 90 and 100 % (3.6 > slope > 3.1) [
14
,
15
].
2. For the reference curve, add plasmid dilutions to a master solu-
tion consisting in 2× SYBR Green Real-Time PCR master mix,
250 nM of HD-V- or GFP-pair primers, in 50
μ
μ
L of total vol-
ume, which is loaded into the 96-well plate.
3. For the samples, add 5
L of undiluted and 1:10, 1:100 dilu-
tions of the extracted vector DNA to the master mix in 50
μ
μ
L
total volume. Samples are amplifi ed in triplicate.
4. The optical 96-well plate is sealed with the optical adhesive
fi lm and spun down 1 min at 4 °C. The reaction is performed
in 7300 Real-Time PCR system under the following condi-
tions: 50 °C for 2 min and 95 °C for 10 min, followed by 40
cycles of 95 °C for 15 s and 60 °C for 1 min.
5. DNA adenovector copy numbers are calculated by replacing
the
y
of reference curve with the Cq values of each sample. The
total number of molecules is determined as follows: number of
vector copies = quantity × dilution factor × 1,000
L,
where the quantity is the number of molecules obtained upon
x
extrapolation from reference curve equation, dilution factor
is the inverse of dilution of the extracted vector detected, and
μ
L/5
μ
