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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 electrophoresis, immediate 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). StaRT-
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TRIS-EDTA based buffer prepared from RNase-free water suitable for storage and dilution of siRNA, pH 8, store at 4 C ...
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