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

p- and down-regulated genes, as well as the houskeeping genes, show similar ratios (Figure 3). The yol 086c and yjl153c ORFs indicate that cDNA labeling and LightCycler quantification (to a higher degree) have a broader dynamic range.

For the genes varying in their expression levels, depending on the target preparation method used (Figure 3D), Light- Cycler analysis confirmed this result. Surprisingly, yor 091w seems to be down-regulated when using the cDNA labeling method, but is up-regulated when applying other methods. This was not expected since cDNA labeling is considered to introduce less bias. Nevertheless, this result was confirmed in an independent experiment (Figure 3E). Besides potential distortion of the relative abundance of individual mRNA by the method used, differences between amplified and non-amplified targets may also be due to different structures (RNA vs. DNA) and length. Targets produced by direct labeling of cDNA varied from 400bp to several kb in length. Amplified targets (cRNA) require fragmentation prior to hybridization.


Therefore those fragments vary from 50bp to several 100bp in length. Whereas 1 g of labeled cDNA could be applied on the array, 10 g cRNA is needed for sufficient signal intensities. Differences in target length, structure (RNA vs. DNA) and concentration can lead to differences based on the formation of secondary structures among different targets during hybridization as well as the variability in hybridization patterns.

Conclusions

The analysis of expression profiles from different organisms and cell types by microarrays is now being implemented in many laboratories. Based on the variable amount of starting material, we have shown that cDNA labeling, linear amplifica
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