Human Unigene RZPD-3 Microarray -
a new tool for functional analysis of the human genome
 

 

Abstract :

Groups of the EMBL and RZPD produced a whole genome cDNA microarray. Based on the NCBI UniGene clustering ( www.ncbi.nlm.nih.gov/Uni-Gene) cDNA clones were selected from the Image clone collection representing 51.145 clusters.
The resulting Human UnigeneSet-RZPD3 was selected according to the following criteria:
- More than one sequence per cluster
- As close as possible to the 3'end of the cDNA
- Length of the insert between 500 and 1200 bp
Of these clones 32.000 have been sequence verified, characterization of further 20.000 clones is in progress. cDNA inserts were PCR amplified, purified, and spotted on glass slides. Analysis of PCR fragments on agarose gels, documentation, control DNAs ("spike-in controls") and Cy5- / Cy3-labeled Oligonucleotides on the slides insure the quality of the microarray and reliability of hybridisation results. Initial comparative hybridisations with RNA from human cell lines demonstrate the potential of the new microarray in biomedical research.

 

 

Production Process of the Microarray
PCR - cDNA inserts were amplified with a pair of NH2-modified flanking universal primers.
Purification - The PCR products were purified on a Biomek FX robot (Beckman) using the Macherey-Nagel Nucleospin kit based on a silica matrix. Since a stacker was added to the robot, 4 x 96well PCR plates could be purified in one run, which takes 90min.
Quality control - The purified fragments were analysed on a buffer free agarose gel system (RTR, Amersham/Pharmacia). Gels were loaded by a Biomek 2000 robot (Beckman). Two gels can be loaded in 30 min, one gel run takes 5 min. 

 

Evaluation and Documentation - Pictures of the Agarose gels were saved as tif-files and analysed with ChipSkipper software, which allows a quantitative (concentration of DNA) and qualitative (multiple or single bands) evaluation of the purified PCR-fragments. The evaluated gel is saved as html-file connected to an access table containing the summary results of all gels analysed. The individual gel pictures can be activated from the summary table.
 
Single bands are quantified according the DNA marker. Missing or multiple bands are marked with red boxes, weak bands with blue boxes.  Yellow boxes mark strong bands with a weaker second band.

Resulting gel pictures are saved as html-files. 

A summary of each gel evaluation is stored in an acess table (with a link to each gelpicture. Bottom line of the table shows the final result of all gels with all PCR products purified.

As a result only 3,95% of the cDNA fragments could not be amplified or showed multiple bands.
Preparation of spotting plates - PCR products were transfered into 384 well microtiter plates in 2xSSC spotting buffer. By using a Biomek FX with a stacker 4 spotting plates could be prepared in one run lasting 40 min.

 

Spotting - spotting is performed on a GeneMachines OmniGrid spotter with 48 split needles on amino-silane coated slides.  One representative of the clone set was spotted on two slides A and B, thus each slide carries more than 26.000 spots including spiking controls and spotted primers. 

 

Slides were scanned with an axon scanner (preview) after spotting and attachment, to control spotting efficiency.

 

 

Complex hybridization
n Isolation of total RNA by standard procedure (Trizol method, PeqLab) from human HeLa and HL-60 cells.
n Direct labeling during cDNA synthesis and purification of probes (Qiagen           
           kit). Here: HeLa sample labeled with Cy3-dUTPs (green), HL-60 
           labeled with Cy5-dUTPs (red).
n Complex hybrization on Human Unigene RZPD-3 Microarray (2 slides,
           about 25.000 spots each): 5xSSC, 0.1% SDS, 42°C, 16h (Amersham
  automated hybridization station).
n Scanning of slides (Axon scanner, 5μm resolution)
n Data analysis with different software tools (GenePix, Axon; ChipSkipper,
           Christian Schwager, EMBL; Spotfire)
 

 

People involved :

Ute Wirkner1, Christian Maercker2, Mechthild Wagner1, Heiko Drzonek1, Uwe Radelof3, Anja Kellermann3, Johannes Maurer3, Christian Schwager1, Bernhard Korn2, Wilhelm Ansorge

 

1        DKFZ
          Deutsche Krebsforschungszentrum, INF360, Heidelberg, Germany
          E-mail: Ansorge@embl.de
                     u.wirkner@dkfz.de
         

 2 :    rzpd

        Deutsches Ressourcenzentrum
        f¨ur Genomforschung GmbH
        E-Mail:info@rzpd.de
           maercker@rzpd.de
        Internet:www.rzpd.de

 

Acknowledgements
n     Jan Selig, EMBL (Robotics)
n        Mona Kessler, Christine Schl¨osser, Christiane Rutenberg,
           Susanne Hermann, RZPD (hybridizations, data analysis)
n        Frank Schwarz, Oliver Heil, Lars Ebert, Peter Neubert, Steffen Schulze-
           Kremer, Martin Holst, RZPD (bioinformatics, poster design)
n        Annemarie Poustka, DKFZ, Hans Lehrach, MPIMG (founder and      
           advisers of RZPD, array design, technology development)
n        Stefan Haas, Martin Vingron, MPIMG (cluster analysis)
n        Martin Stock, RZPD (administrative managing director)
n        BMBF (financial support)