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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.* (www.genexnat.com). Reagents 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 G.E.N.E., Inc.
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 City, CA).
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