Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR
matrix and the remaining
wells were loaded with 5 l each
of molecular weight marker. One
microliter of DNA ladder was
loaded into a ladder well and 1 l
of PCR product was loaded into
each sample well. The chip was
vortexed and placed into the Agilent
2100 bioanalyzer. DNA 7500
assay was run, which applies a
current sequentially to each sample
to separate products. DNA is
detected by fluorescence of the
intercalating dye in the gel-dye
matrix. NT/CT ratios were calculated
from the area under the
curve for each PCR product and a
size correction was made since an
intercalating dye was used to
detect DNA. The area under the
curve values for each NT and CT
was entered into the G.E.N.E., Inc.
spread sheet. Alternatively, the
concentration values, which are
based on the areas, can be used to
calculate the NT/CT ratio.
Agarose gel electrophoresis
Following amplification in the
Rapidcycler, PCR products were
loaded directly onto 4 % agarose
gels (3:1 NuSieve: SeaKem) containing
0.5 g/ml ethidium bromide.
Gels were electrophoresed
for approximately one hour at
225 V and visualized with a
Foto/Eclipse image analysis system
(Fotodyne, Hartland, WI). Digital
images were saved on a Power
Mac 7100/66 computer and Collage
software (Fotodyne) was
employed for densitometric analysis.
Quantification of gene expression
was determined according to the
G.E.N.E., Inc. manual. These
mathematical steps are programmed
as formulas into a
spread sheet available directly or
downloadable from G.E.N.E., Inc.
so that only the raw data values
for each NT and CT for each gene
must be entered. The gene expression
values then are calculated
PCR products amplified with fluorescent
primers in the PTC-100
block thermal cycler were det
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