Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR
ed. The intensity of
the bands, which is used for the
calculation of the gene expression,
is determined by appropriate software
from the gel image. The Agilent
2100 bioanalyzer obtains signals
in digital format. The electrophoretic
traces of 12 samples
are shown in figure 1 B. Alternatively,
these data can also be displayed
as a gel like image. The
ABI 310 Genetic Analyzer also
acquires digital data. Figure 1 C
shows several electropherograms
from pooled PCR reactions. In
each case, NT bands appear above
CT bands. Quantification of gene
expression was determined
according to G.E.N.E., Inc. protocol.
The formula calculating gene expression in the form of molecules/
106 molecules was incorporated
in an MS Excel file. Each
gene was measured with each
method. Genes measured by all
three methods in figure 1 are -
actin, CDC2, E2F1, E2F4, p18 and
TNF receptor. No statistically significant
differences between values
obtained by the three methods
were detected for 14/15
genes. Gene expression values
for one gene, E2F5, obtained by
each electrophoresis method
were significantly different from
each other. The most likely reason
that E2F5 values differed
between methods is that the CV
for each method was so low. If
the coefficient of variance (CV)
for each method (agarose gel,
ABI Prism 310, Agilent 2100 bioanalyzer)
had been at the average
level for each method , there
would not have been a significant
difference in the means measured
for E2F5 (figure 2 on
Three or more replicate measurements
were made for 44/45 gene
expression values presented in figure
2. Reproducibility was best
with the Agilent 2100 bioanalyzer
(CV = 0.26). The results for
agarose gels are similar to those
reported for other studies in
which standardized, quantitative
RT-PCR was used.
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