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ĄComparing with microarray hybridization system, two fluorescent dyes such as Cy3 and Cy5 are usually used in the microarray experiment.
That is because two targets at one hybridization is thought to reduce the error during the hybridization and make the normalization between hybridization experiments easier. We agree to accept this advantage, however,the fluorescent levels of Cy3 and Cy5 dyes after scanning is actually so different. In the case of Type 7 slide glass which was metal coated for the enhancement of Cy5 signal, the level of Cy3 signal is no so high as the level of Cy5. Therefore the adjust of both signal seems hard. This reason and one more reason such as the comparison of more than three target data, we prefer to use single target system. Normally we have labeled the target RNA with Cy5-dCTP. Normalization of the data among different glasses were made by the signal of positive control clones or the sum of the whole probe signals.
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