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Start of funding 01.01.2001

Development of disease-specific cDNA arrays

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Early diagnosis is essential for prevention and treatment of neoplastic as well as for severe disabling diseases. As markers for these phases are lacking, the intention of the project is to use a chip-based multi-gene cDNA array to establish a disease specific array to facilitate a novel molecular approach. In detail, a member of our research group will learn and perform the recently developed multi-gene array technique under Dr. Michael McClellands supervision at the Sidney Kimmel Cancer Center (La Jolla, CA, USA) in a pilot study with selected tissue samples from patients with colon carcinoma and rheumatoid arthritis to be able to transfer the technique to our research group and, vice versa, to refine Dr. McClellands technology with samples of hitherto not evaluated diseases. The overall goal of this study is to provide the basis for a novel gene detection system for diagnosis of premalignant stages of neoplastic diseases or early inflammatory stages of disabling diseases.

Final report:
The aim of the project was to develop so-called „vertical microarrays“ that allow the expression analysis of up to a few dozen genes in hundreds to thousands of samples, as opposed to conventional “horizontal” microarrays that can examine the expression of thousands of genes in only a few samples. Especially in complex diseases such as rheumatoid arthritis (RA), it is not enough to analyze only a few patient samples to get an adequate insight into the disease-specific gene expression. On the other hand, in RA research there exists already a pool of genes of interest, of which their expression should be tested in a larger patient group - a global expression analysis is not absolutely necessary.

In theory, the easiest way to create such an array would be to just spot one cDNA sample per patient on a glass slide. Unfortunately, this sample would be far too complex, consisting of thousands of different sequences, so that each sequence is not represented often enough to be detected by a hybridization probe.

To reduce the complexity of the samples and at the same time amplify the cDNA while maintaining the relative expession ratios, we used a method developed by Profs. Welsh and McClelland called RAP-PCR (1) (RNA arbitrarily primed polymerase chain reaction). This technique allows the amplification of subgroups of the available cDNA using short arbitrary primers.

The goal of the part of the project, which was sponsored by BaCaTec, was to establish the basic parameters for thid technique, so that it could thereafter be expanded to include a broader patient group. For this purpose cDNA from 20 patients was amplified using 23 different primers and spotted on aminosilane-coated glass slides using a spotting robot. Then fluorescence-labeled probes were synthesized for several genes of interest. This was first done by PCR, but these doublestranded probes revealed to be unsuitable for our purpose. We then altered the protocol and continued by producing single-stranded RNA probes by in vitro transcription, and this led to much better hybridization signals. These probes were then hybridized to the arrays to detect the corresponding genes.

During the supported period it could be shown that the method is feasible and delivers the expected results. The method was further optimized and adapted to a high throughput aproach during a two-year stay in San Diego, again by Dr. Martin Judex, that was supported by a grant of the “Deutsche Akademie der Naturforscher Leopoldina”.

Literature:

(1) Rondeau G, McClelland M, Nguyen T, Risques R, Wang Y, Judex M, Cho AH, Welsh J. Enhanced microarray performance using low complexity representations of the transcriptome. Nucleic Acids Res. 2005 Jun 24;33(11)

A second publication is in preparation.