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Gene Expression Arrays:,,,Highly Sensitive Detection of Expression Patterns,,,with Improved Tools for Target Amplification

f ≤0.5 in the direct-labeling approach. These expression ratios were very reproducible for each target-labeling method applied. This is shown in Table 1 for the cDNA labeling procedure. The vast majority of the genes (97%) showed a less than twofold variation from the average expression ratio. Similar results can be obtained by comparing linear amplification with linear amplification, and random amplification with random amplification (data not shown).

The most important question is whether the three applied methods deliver similiar expression differences. Therefore we compared the cDNA labeling method with the amplification methods. Table 1 shows that 92% of the yeast genes show comparable expression alterations (≤ 2-fold variation) when cDNA labeling is compared to linear amplification. When cDNA labeling is compared with random PCR amplification, 89% of the yeast genes show similar expression rates. These results indicate that different amplification methods might not generate the same number of labeled target molecules from each template. Rather, the degree of amplification within a method is reproducible from one reaction to another. In addition, the different methods deliver similar expression alterations.

Validation of results with quantitative PCR on the LightCycler Instrument
To find out which target preparation methods would reflect the biological situation best, an independent method (quantitative RT-PCR) was applied to a number of genes. We randomly selected two genes from a group of upregulated genes and two genes from a group of downregulated genes in rich vs. minimal medium. We also selected three housekeeping genes that should show no alterations, and four genes from the group in which different methods show differences in the expression alterations. The group of u
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