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 chan- nel, and allow quantitative gene expression measurement at the end- point of PCR, without real-time analysis. Gene expression measure- ment of 15 genes was more reproducible with the Agilent 2100 bioana- lyzer than with the ABI Prism 310 Genetic Analyzer or agarose gel elec- trophoresis methods. The mean coefficient of variance (CV) for these three methods was 0.26, 0.39 and 0.50, respectively.
The document details how the Agilent 2100 Bioanalyzer can be used in combination with Standardized RT (StaRT)-PCR reagents from Gene Express National Enterprises to obtain highly reproducible, standardized gene expression measurements. The internal standards used in StaRT-PCR control for variable loading of PCR products into electrophoresis channels.
The study compares the reproducibility of this method against agarose gel electrophoresis and the ABI Prism 310 Genetic Analyzer. Results indicate that gene expression measurement of 15 genes was more reproducible with the Agilent 2100 bioanalyzer, showing a mean coefficient of variance (CV) of 0.26 compared to 0.39 for the ABI Prism 310 and 0.50 for agarose gel methods.
The mean CV was 0.26, which was lower than the ABI Prism 310 Genetic Analyzer (0.39) and agarose gel electrophoresis methods (0.50).
The method uses Standardized RT (StaRT)-PCR reagents from Gene Express National Enterprises.