Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR
This application note describes how the Agilent 2100 bioanalyzer can
be used in combination with Standardized RT (StaRT)-PCR reagents
from Gene Express National Enterprises (G.E.N.E.), Inc. (Huntsville,
AL, USA) to obtain highly reproducible, standardized gene expression
measurement with RT-PCR. The internal standards used in StaRT-PCR
control for variable loading of PCR products into electrophoresis channel,
and allow quantitative gene expression measurement at the endpoint
of PCR, without real-time analysis. Gene expression measurement
of 15 genes was more reproducible with the Agilent 2100 bioanalyzer
than with the ABI Prism 310 Genetic Analyzer or agarose gel electrophoresis
methods. The mean coefficient of variance (CV) for these
three methods was 0.26, 0.39 and 0.50, respectively.
We combined the Agilent 2100 bioanalyzer
with a quantitative
method for standardizing gene
expression, Standardized RT
(StaRT)-PCR (Willey et al, Am. J.
Resp. Cell Mol. Biol., 19, 6, 1998),
to produce a sensitive, reproducible
method for quantitative
end-point PCR measurement of
gene expression. The Agilent 2100
system markedly simplifies electrophoresis.
There is no need to
prepare agarose gels and, following
results are provided by PCR product
quantification software. The
internal standards used in StaRTPCR
control for variable loading
of PCR products into electrophoresis
channels, and allow
quantitative gene expression measurement
at the end point of PCR,
without real-time analysis. StaRTPCR
used with agarose gel electrophoresis
has been validated in
independent studies (Loitsch et al,
Clinical Chemistry, 45, 619, 1999;
Allen et al, Am. J. Resp. Cell Mol.
Biol., 21, 693, 1999; Mollerup et al,
Cancer Research, 59, 3317, 1999).
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