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Quantitative end-point RT-PCR gene,,,expression measurement using the,,,Agilent 2100 Bioanalyzer and,,,standardized RT-PCR

ed. The intensity of the bands, which is used for the calculation of the gene expression, is determined by appropriate software from the gel image. The Agilent 2100 bioanalyzer obtains signals in digital format. The electrophoretic traces of 12 samples are shown in figure 1 B. Alternatively, these data can also be displayed as a gel like image. The ABI 310 Genetic Analyzer also acquires digital data. Figure 1 C shows several electropherograms from pooled PCR reactions. In each case, NT bands appear above CT bands. Quantification of gene expression was determined according to G.E.N.E., Inc. protocol. The formula calculating gene expression in the form of molecules/ 106 molecules was incorporated in an MS Excel file. Each gene was measured with each method. Genes measured by all three methods in figure 1 are - actin, CDC2, E2F1, E2F4, p18 and TNF receptor. No statistically significant differences between values obtained by the three methods were detected for 14/15 genes. Gene expression values for one gene, E2F5, obtained by each electrophoresis method were significantly different from each other. The most likely reason that E2F5 values differed between methods is that the CV for each method was so low. If the coefficient of variance (CV) for each method (agarose gel, ABI Prism 310, Agilent 2100 bioanalyzer) had been at the average level for each method , there would not have been a significant difference in the means measured for E2F5 (figure 2 on page 6).

Reproducibility
Three or more replicate measurements were made for 44/45 gene expression values presented in figure 2. Reproducibility was best with the Agilent 2100 bioanalyzer (CV = 0.26). The results for agarose gels are similar to those reported for other studies in which standardized, quantitative RT-PCR was used.'"/>

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