Project organisation and collaboration

By high throughput robotic techniques, tens of thousands of small spots of gene probes are placed on glass slides, which are used to measure gene activity on a genomic scale. This technique is a quantum leap in the study of transcriptional changes in tumours (and in disease in general), as well as for experimental studies, and probably will revolutionise the differential diagnostics of tumours, paving the ground for much more biologically founded therapy selection.

Three main strategies have been followed: Acquiring shared user equipment, centralised in a core facility with dedicated personnel. Establishing a production facility enabling DNR groups to design any contact-printable array type. Stimulating competence sharing through weekly project meetings, open to all DNR groups. The considerable resources needed for the core facility and personnel has been obtained by an offer to also serve the entire Norwegian scientific community, and this has allowed us to build competence at DNR in both experimental techniques and related bioinformatics. This again has put DNR in the research focus, and thus benefits our strategic research goals in general. Our networking activities have led to strategic alliances also in bioinformatics, e.g. as main bioinformatics collaborator of the Norwegian Computing Centre, and initiatives by Eivind Hovig have led to a central role in the FUGE-related bioinformatics network in the Oslo region.

Initially there was strong competition with groups at the National and Ullevål Hospitals who wanted to submit competing platform applications to FUGE, based on the commercial Affymetríx technology. However, this was successfully merged with the NMC, and is now running as an NMC-associated Core Facility, with some funding from NMC. Clearly, this voluntary competition is important for confidence in our services; if people prefer that technology, we provide it, and if we cannot compete, we will discontinue the spotted array service. So far, both technologies are in high demand, and Affymetríx technology is available to DNR groups.

The main task of DNR is to produce human arrays, and at present we provide cDNA-based arrays with 18 000 probes. The DNR groups have been allowed to include additional probes of particular interest to their projects. Although we have 46 000 cDNA clones altogether, we have halted the extension of the cDNA arrays in antecipation of a move to oligonucleotide arrays. The NMC has purchased a set of 26 000 oligonucleotide probes for expression analysis in man, but as it now appears that a better set, based on the Ensembl database rather than Unigene is available, production has been postponed unitil negotiations about exchanging this are completed. Hopefully we will be able to provide arrays of 37 000 Ensembl-based probes sometime after October.

After continuously producing good arrays, we had several months of production problems last fall. The problems came together with experimental problems caused by variation in the quality of the fluorescent dyes, and thus several batches were produced, and some distributed, before we realised we had a problem also with the slides. On more than one occasion, testbatches indicated that we had regained quality after changing some parameters, but scaling up and enforcing production to satisfy demands resulted in a series of useless full batches. In the end, installing complete climate control in the (temporary) printing room solved the problem. All users had their slide batches from this period replaced free of charge (about 200 slides), and about 1000 slides were discarded by our internal QC. Although this was expensive for a non-profit producer, more important was the possible effects on confidence. The resulting delays in experiments for several groups clearly was very unpleasant, but it is our impression that although it took longer than one would wish, we handled the problems reasonably well, and do not seem to have lost users. During this period, som users tried commercial slides, but most have now come back to our slides. On the other hand, we have negotiated about 30% price reductions on slides from Agilent, to always have an altenative for people using spotted glass arrays.

The technology for design, production and use of genomic microarrays, made by spotting representations of large genomic clones (BACs or PACs) rather than cDNAs, has been established by the Myklebost group (Leonardo Meza-Zepeda), and is now used to produce a global human genomic array with about 1 Mb coverage. Whereas we have a number of global BAC collections in house, the production of probes from these is very resource-demanding (entailing 24 000 PCRs for the present 4000 probe set), and among the global sets we have in-house, we have chosen to concentrate on a set from the Sanger Centre, selected from the genome project sequenceing path, so that the sequence content and position is known. This greatly facilitates down-stream analysis, such as supplementation of intervening probes and gene validation. This array will be provided as a general resource at a non-profit price. The same group, together with Junbai Wang, has developed software taking care of the special requirements of analysis of genomic array results.

A very interesting new technology is equipment to provide on-the-fly custom oligo-nucleotide arrays, such as the of the German Febit company. The NMC is evaluating such possibilities, which would be very resource-demanding on the bioinformatics side, but has exciting applications for our users, such as detailed mapping of amplifications and deletions detected with genomic arrays, large-scale scoring of exon expression and splicing, methylation and chromatin immunoprecipitation (”ChIP-chip”) analysis etc.
Some DNR groups have a need for arrays from other organisms, such as E. coli and S. pombe, but the project has not had the resources to provide probe collections for single groups. In such cases, the formation of probe collection consortia with other groups in the field, or the use of Affymetrix arrays when such are available, has been recommended.

Whereas the FUGE platform has been of major strategic importance, and at present supports our activities, it also makes some demands. To get the resources, we have (together with our NMC partners) offered to provide services, bioinformatics resources, give courses, and produce custom arrays for any Norwegian project, not only within our own field of interest. Space is very precious, and the project consumes a lot of scientific leadership resources (which has been somewhat alleviated by the employment through FUGE of a 20% assistant project leader, Leonardo Meza-Zepeda).

The core facility, in addition to the array-printing and liquid-handling robots, has two array scanners, an automatic hybridisation station (a second will arrive shortly), a Nanodrop spectrophotometer, and an Agilent Bioanalyzer, primarily for local users. The equipment is to a large extent funded by Dnr, and is cared for by the core facility personnel. A local implementation of the BASE microarray database system (designed in Lund, and further developed in collaboration with them) serves the entire region with structured storage of array data and experiment information, as well as some analysis tools. This solution is now implemented also in Bergen and Trondheim. Through the NMC, all users get free licences for the J-express Microarray Analysis Package (clustering etc), and work is in progress to integrate it further with BASE.

A large fraction of the local applications of microarrays are of translational nature, in that the clinically relevant properties of tumour samples or cell lines are investigated, such as chemotherapy resistance, radiation response, micrometastatic cells, differential diagnostics, etc. Through scientific collaborations with leading international groups, DNR has also acquired a high level of competence in the necessary experimental techniques, and is in the process of developing a critical level of bioinformatics competence. The local project has a weekly meeting, organised and led by the core facility personnel and open to anyone from DNR, where project-related questions are discussed. This contributes to the institutional teamwork spirit that has been instrumental in the establishment of all the necessary techniques, equipment and competences needed. About 10 DNR groups are currently engaged in microarray studies, and these have consumed about half of the output of human cDNA slides (average 1000+/year 2001-3). In addition to the provision of facilities, infrastructure and user support, the priority area has supported local projects through free arrays up to last year (when DNR funded a large part of the Core facility), and by a 75% subsidy from this year (as we now have to pay full price for the arrays).

The project also supports the development of protein array techniques by the Hovig and Sioud groups by our custom production services.