Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Gene expression profiles of different cell lines based on the technology of Clondiag

Gene expression profiles from various cell lines were generated. Gene expression profiling for pharmacological testing was optimised. For this, experiments were carried out that should lead to an adaptation of the Clondiag AT-platform (a tool developed for genotyping) for use in gene expression profiling. The AT-platform is limited in the number of genes that can be analysed, but first experiments indicate, that a much higher sensitivity may be possible.

We optimised the experimental conditions for the analysis of lower abundant expressed genes (low amounts of gene specific mRNA) and optimised the proprietry array tube system for gene expression profiling. For this optimisation we focused on 50 human genes which are intensively studied at the Klinik für Innere Medizin (University Jena) and for which reference clones are available. A major focus of the work was to optimise the algorithm for the calculation of oligonucleotide probes that are most promising for gene expression analysis. This includes secondary structure prodictation, information about heterolougous sequence hybridization and exclusion of repeat and repeat like sequences.

We also assayed the quality of oligonucleotide synthesis, which has a significant impact on the specificity, and sensitivity of the system.

The most important improvement for the quality of the assay is our new optimised labelling protocol. Unfortunately, until now we could not fully confirm, that the quantitative results obtained give a true quantitative reflection on the expression of the respective genes. This problem however is to date not solved with any array based method. (Only RNAse protection assays and with slightly lower precision real time PCR allow to calculate absolute number of nucleic acid targets.)

Reference experiments with rectal biopsies from healthy donors allowed us to establish an optimal homogenisation protocol of unfrozen tissue that can be combined with the Qiagen RNA extraction method.

Gene expression profiling from biopsies with and without electrophysiological measurements (in cooperation with Partner in Hannover), showed that no significant change could be observed when samples are analysed in an Using Chamber before RNA extraction. This does not exclude that "sensitive" genes exhibit changes in gene expression upon preanalysis treatment.

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Contacto

Eugen ERMANTRAUT, (CEO)
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