Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR
by capillary electrophoresis in
an ABI Prism 310 Genetic Analyzer
under denaturing conditions.
One microliter of each PCR reaction
or 1 ml of a mixture of PCR
reactions was combined with 9 ml
formamide and 0.51 ml of ROX
1000 size marker. Samples were
then heated to 94 C for 5 minutes and flash-cooled in an ice slurry.
Samples were loaded onto the
machine and electrophoresed at
15 kV, 60 C for 35-45 minutes
using POP4 polymer and filter set
D. The default injection parameters
of 15 kV, 5 seconds were
used. The fragment analysis software
GeneScan (Applied Biosystems,
Inc., Foster City, CA,) was
used to determine peak sizes in
base pairs and peak heights which
were used to calculate NT/CT
ratios. No size correction was
performed since each DNA molecule
was tagged with one fluorescent
marker from one labeled
primer. The area under the curve
values for each NT and CT was
used for calculations.
All statistical analyses were conducted
using SAS version 6.11
(SAS Institute, Cary, NC). A GLM
test followed by Duncan analysis
was used to test for significant differences
in gene expression values
obtained by the three methods of
detection within the same cDNA.
Differences were considered statistically
significant if the P value
was less than 0.05.
Comparison of three methods
Gene expression values for 15
genes were obtained by three
methods of electrophoresis from
the same cDNA, as described. Figure
1 on page 5 depicts representative
results obtained by each of
the three electrophoresis methods.
The agarose gel shows the familiar
band pattern (figure 1A). Although
some of the bands are distorted a
good resolution and quantification
can be obtain
Source:Page: All 1 2 3 4 5 6 7 Related biology technology :1
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