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Sonderforschungsbereich 415:
"Specifity and Pathophysiology of Signal Transduction Pathways"
at Christian-Albrechts-University in Kiel
Project Z1: »Systematic expression analysis«
Summary
The pattern of genes expressed in a cell can provide important information about the cell state. cDNA microarray technology can measure the expression of thousands of genes in a biological sample. These microarrays have been used increasingly in the last few years and have the potential to help advance our biological knowledge at a genomic level.
Among the different expression analysis systems, glass based microarrays represent a powerful and cost-effective tool for quantifying gene transcription.
The mucosal immunology group (MIG) have developed a new generation of glass-based cDNA microarrays as part of the SFB 415. This collaboration of research groups at the university of Kiel was established to design the microarrays. The aim was to produce four different topic-related microarrays:
- apoptosis
- differentiation and regeneration
- inflammation and infection
- murine array.
The members of the SFB 415 selected genes of interest for each topic.
The MIG used gene-specific PCR-products representing only well characterized genes. Bioinformatic methods were used to design the primers in order to ensure a high specificity of the PCR-products and homogenous hybridization conditions for the resulting microarray.
The cDNA microarrays were produced in collaboration with Holger Eickhoff (Scienion, Berlin.
A typical microarray experiment starts with the extraction of the RNA, the labelling with fluorescent dyes followed by the hybridization. After hybridization, microarray gridding is carried out using AIDA (Raytest). We have established a Laboratory Information Management System (LIMS), which encompasses all significant data (sample information, experimental conditions, microarray raw data etc.) relating to microarray analysis. The LIMS user interfaces allows the recording and easy access to data at all stages of the microarray experiment process. After data normalization, a LIMS user interface has been generated to provide data analysis and visualisation tools, to allow interpretation of the large number of data points generated by expression screening. Differences can be evaluated using cluster analysis, parametric and non-parametric statistics. Many of the tools can be made available on the internet, so that data can be easily exchanged between the participants of the SFB.
The verification of significant results will be carried out by using real-time PCR analysis. The advantages of TaqMan compared to conventional PCR techniques are high throughput and reproducibility, with low noise variability.
The establishment of an expression profiling platform including the development of new microarrays will provide a powerful tool for elucidating functional genomics in chronic diseases.
- Custom-made arrays will only contain genes of interest to the various groups, thus eliminating a large degree of redundancy in the data generated, when compared to using pre-made commercial arrays.
- The new quality standards of the cDNA microarrays will result in more reliable data compared to traditional clone-based cDNA microarrays.
- The generation of the same quality assured arrays and storage of data in a central database will promote easy data sharing and collaboration between the participants of the SFB 415.
- The central expression profiling facility will provide a variety of analysis tools to handle the large quantity of data generated by expression screening experiments.
Original papers
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Costello, C. M., Mah, N., Hahn, A., Lu, T., Albrecht, M., Gurbuz, Y., Nikolaus, S., Hampe, J., Klöppel, G., Eickhoff, H., Lehrach, H., Lengauer, T., Schreiber, S. (2004)
 Dissection of inflammatory bowel disease pathophysiology using high-density full-genome cDNA microarrays |
| PNAS, in revision |
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Lu, T., Costello, C.M., Croucher, P.J.P., Häsler, R., Deuschl, G., Schreiber, S.. (2004)
A simple and general microarray normalization technique using Zipf's law. |
| Nucleic Acids Research, in Revision |
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Mah, N., Thelin, A., Lu, T., Nikolaus, S., Kühbacher, T., Gurbuz, Y., Eickhoff, H., Klöppel, G., Lehrach, H., Mellgård, B., Costello, C. M., Schreiber, S. (2004)
Comparison of oligonucleotide and cDNA-based microarray systems |
| Physiological Genomics, in press |
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