Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 144

3. Prepare sufficient PCR master mix for triplicates of each plasmid
standard, vector DNA sample, and NTC. A single 10 μL PCR
master mix is comprised of 6.25 μL of 2× SYBR Green master
mix, 0.5 μL of 5 μM WPRE primer mix, and 3.25 μL PCR
grade H 2 O ( see Note 7 ).
4. Set up three replicate PCR reactions (total volume 12.5 μL),
comprising of 10 μL of PCR master mix and 2.5 μL of DNA
sample, for each standard, vector sample or NTC in wells of a
384-well optical reaction plate, and seal the plate with an opti-
cal adhesive cover. Protect the plate from light by wrapping in
aluminum foil and store at 4 °C until time of run.
5. Centrifuge the plate for 2 min at room temperature at 1,700 × g ,
and transfer the plate to a real-time thermal cycler (we use the
Applied Biosystem 7900HT real-time PCR system) and run
real-time PCR using the following cycling conditions:

Real-time amplification Step 1 (1 cycle) 2 min 50 °C
10 min 95 °C
Step 2 (40 cycles) 15 s 95 °C
1 min 60 °C

Melting curve analysis Step 3 (1 cycle) 15 s 95 °C

15 s 60 °C

1. Follow the instrument instructions to evaluate the amplifica-
tion curves generated for standards and samples. If required,
manually adjust the baseline and threshold settings. Check the
amplification curves of each set of three replicates and omit any
wells that show amplification variation. If an entire set of three
replicates varies greatly from the other two sets for a particular
vector sample, omit this set of data from the analysis. Evaluate
the standard curve of Ct versus log input copies of standard
plasmid. Check that the slope of the standard curve is close to
3.2 and that the correlation coefficient ( R 2 value) is 0.99; oth-
erwise repeat the run with a new set of standards.
2. Export the data into an Excel spreadsheet. Determine the
mean quantity (starting copies per reaction) for all wells of an
unknown vector sample (total of 9 values if none of the values
have been omitted). Calculate the genomic titer (vector
genomes (vg)/mL) of the original vector stock using the fol-
lowing equation:

3.3.3 Data Analysis

DNase esistant vector genomesper mL
mean quantity of target a

-
= ´ ´ bbc ´

a , dilution factor = 5,000; b , factor for conversion for copies
per 2.5 μL to copies per mL = 400; c , conversion factor for
estimation of a double-stranded (plasmid) template to a single-
stranded (AAV) template = 2