The development of novel pharmaceutically active compounds requires effective methods in order to increase throughput and focus corporate research and development. The increasing number of potential compounds in pharmaceutical research and the shortened time frame in the drug development have created a need for novel methods in order to accelerate development processes. Among other methods, such as Real-Time RT-PCR, gene expression analysis with customized cDNA chips providing study relevant markers have shown to be a valuable tool for surrogate markers identification and monitoring. The present paper describes the preparation and application of cDNA chips for apoptosis targets genes as well as for calbindin D-28k and cytokines. Validation of the results are performed using the Real-Time RT-PCR technology. In addition, investigations regarding the requirement of replicates as well as the development of tools for chip quality control are discussed.Results of an in-house cDNA mid-density feasibility study are shown. Customized multiple use chips with genes for apoptosis, cytokines, and CYP450 were prepared. The chip preparation steps such as ink-jet printing, incubation and baking were monitored with Differential Interference Contrast microscopy (DIC) which gives information on e.g. missing spots, overlap, and homogeneity and can therefore be used as tool for quality control. The correlation between number of replicates and coefficient of variation (CV) of the fold change values was investigated with specially designed chips and ink-jet and pin spotter principle was compared. In addition, direct determination of the background and background extrapolation was compared and the impact on the obtained fold change values was investigated and discussed with regard to non-specific binding of labeled species in the sample solution. The customized cDNA chips were used in studies within pharma drug development and the results were compared to RT-PCR.
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