Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR
PCR reagents for over 400
genes are now available commercially
through Gene Express
National Enterprises, Inc.*
for an additional 2000 genes are in
production. In a blinded inter-laboratory
study of common samples,
this method provided excellent
reproducibility (manuscript in
preparation). Thus, all laboratories
using a common mixture of
internal standards for StaRT-PCR
will be able to enter gene expression
data into a common gene
expression databank. Such a databank
has been established at
StaRT-PCR reactions include
cDNA from a cell or tissue sample
of interest and a standardized mixture
of competitive template internal
controls for each gene to be
measured. Quantitation is based
on a ratio of each endogenous
gene (referred to as native template
[NT]) PCR product to the
corresponding shorter internal
standard competitive template
(CT) product. Until recently, the
primary method used to size, separate
and detect NT and CT StaRTPCR
products has been agarose
gel electrophoresis. This method
is available to most laboratories
without the purchase of additional
equipment. However, recently
available alternative methods for
electrophoresis, including the Agilent
2100 bioanalyzer have the
potential to markedly increase
throughput and reproducibility.
Additionally, if StaRT-PCR is to
have broad application, it is
important to determine whether
electrophoresis and analysis of StaRT-PCR products with different
instruments will provide comparable
data. This question was
addressed by comparing StaRTPCR
expression data for 15 genes
obtained following electrophoretic
separation of PCR products by
either the usual agarose gel, the
Agilent 2100 bioanalyzer (Agilent
Technologies, Inc., Palo Alto, CA),
or the ABI Prism 310 Genetic Analyzer
(Applied Biosystems, Foster
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