ArrayNL platform^©

ArrayNL - first meeting

First ArrayNL meeting

PROGRAM
13.30-14.00	Welcome / Coffee
14.00-14.30	Genome-wide analysis of transcription circuitry (Dr. Frank 
		Holstege - University Medical Center Utrecht)
14.30-14.50	Determing expression patterns in fruit development (Oscar 
		Vorst - CPRO-DLO, Wageningen)
14.50-15.45	DNA-chips and arrays in NL; equipment, state of the art, 
		planned applications
	14.50	Amsterdam-AMC:	Marian Groot Koerkamp - "DNA-array technology
                at the AMC"
	15.05	Amsterdam-NKI:	Ron Kerkhoven
	15.15	Leiden:		Ton Raap - "Implementation of array technology
		in the LUMC"
	15.30	Maastricht:	Frank v.d. Loop - "Microarray technology in
		Maastricht: Towards GenomeCenter Maastricht"
	15.45	Utrecht:	Frank Holstege
16.00-16.15	Project decriptions: Peter Groot / Martien Groenen (5-10 
		min/speaker)
	16.15	General discussion (local plans, national desires [clone 
		collections, protocols], next meeting, etc.)
16.30-..... Close / Drinks

Genome-wide analysis of transcription circuitry

Frank Holstege

Organisms respond to environmental changes as well as to internal programs of growth and development by changing their patterns of gene expression. In order to understand the regulatory circuitry of a eukaryotic genome, genome-wide expression analysis in yeast was used to identify genes whose expression depends on the functions of key, conserved components of the transcription initiation machinery such as subunits of the RNA polymerase II holoenzyme, TFIID, SAGA and nucleosomes. The talk will focus on the technology and data analysis used. The results that will be discussed include the following. The requirement for Srb4, a component of the RNA polymerase II holoenzyme, was indistinguishable from the requirement for core RNA polymerase II. This implies that an Srb4-containing holoenzyme is involved in the transcription of all protein-coding genes. In contrast, other components of the RNA polymerase II holoenzyme, as well as components of the general transcription factor TFIID were found to have roles at only distinct sets of genes. Comparison with genome-wide expression profiles obtained during the mitotic cell-cycle, sporulation and nutrient starvation, indicate the involvement of specific components of the transcription machinery in these processes. Comparison between the sets of genes regulated by diverse factors indicate functional interactions. Depletion of histone H4 reveals the role of nucleosomes in transcription as well as in telomeric silencing. These results, as well as other insights gained from genome-wide expression profiling will be presented.

Dr. Frank C.P. Holstege, Head Genomics Laboratory Department for Medical Genetics University Medical Center Utrecht, KC.04.084.2, PO Box 85090, 3508 AB Utrecht, The Netherlands
E-mail: f.c.p.holstege@med.uu.nl Tel: +31-30-2503822/ 2503800 Fax: +31-30-2503801/ 2505301

DNA-array technology at the AMC

Marian Groot Koerkamp

A facility to address DNA-array technology is being set up at the AMC. Since there are many groups with different types of application for this technique, a flexible approach is being taken. Our arrayer is home-built and can spot with different types of printheads nto filters as well as glass (different needles are used for the two applications).

For the glass arraying several binding protocols will be investigated. At this moment, our group has most experience with the "macro-array" yeast genome filters, provided by dr. J. Hoheisel from the DKFZ in Heidelberg. Filters are used with 33P-labeled cDNA probes and can be re-used easily. The results are scanned on a Storm 860 imager; image files are loaded in the Visage HDG-Analyzer program (Bioimage) to run a spot-finding algoritm and quantification. A platform-independent web-interface is being set up to normalize and interrogate resulting analyses-output files.

Department of Biochemistry, University of Amsterdam Academic Medical Center Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Tel: +31-20-5665130/5665381 Fax: +31-20-6915519 E-mail: m.koerkamp@amc.uva.nl

cDNA microarray hybridization at the Leiden University Medical Center

Ton Raap, Johan den Dunnen, Marlies van de Berg, Victor de Jager, Frans van de Rijke, Hans Tanke and GertJan van Ommen. Departments of Human Genetics and Molecular Cell Biology, Leiden University Medical Center

At the Leiden University Medical Center, the Departments of Human Genetics and Molecular Cell Biology are in the process of implementation of cDNA microarray hybridization technology.

The arraying hardware currently features a Genetic Microsystem Arrayer. It uses a pin and ring system and high speed robotics for creating spots with 150 um diameter and 250 um center-to-center distance. The system accommodates three microtitre plates and a maximum of 42 microscopic object slides.

A single 96 well plate is copied to the 42 slides in about 1 hr. This period of time includes the rinsing and drying of pins. The arrayer's software supports a 96 well microtitre plate format, with an upgrade to 384 well format expected this summer. In the 4 months period of use, the system proved reliable.

The scanning hardware features since two months a Genetic Microsystem Scanner which employs a flying objective to scan a full microscopic slide at 10 um resolution in 4 min, with the epi-illumination sources being solid state lasers (532 and 635 nm). Results are exportable as TIFF files and are analyzed -for the time being- with the Scanalyze Software freely available from Pat Brown's Lab. The sensitivity of the system in terms of number of Cy3 molecules detectable per spot has been determined by us to be 10⁵ molecules (600 per pixel.or 6 per um²)

With the essential hard- and software conditions for cDNA microarray hybridization experiments fulfilled, the "wetware" aspects of microarraying are now under study: cloned cDNA/EST probe generation, immobilization substrates, probe immobilization chemistries, cDNA synthesis/amplification and labeling, hybridization and detection/signal amplification conditions.

Microarray technology in Maastricht: Towards GenomeCenter Maastricht

HJM Smeets (Bert), FTL van der Loop (Frank), JPM Geraedts (Joep)

The GenomeCenter Maastricht is a joint initiative of the Division of Genetics1, the Research Institute for Growth & Development (GROW)2, the Cardiovascular Research Institute Maastricht (CARIM)3, the Nutrition Toxicology and Environment Research Institute Maastricht (NUTRIM)4 and the Research Institute for Extramural and Transmural Health Care (ExTra)5.

Microarray technology at the Maastricht University, the Research Institutes, and the Academic Hospital Maastricht is initiated and coordinated by the Division of Genetics. Our mission statement is to provide the infrastructure for working with microarrays, and to develop standardized protocols and custom-made microarrays for gene expression studies, mutation analysis and polymorphism studies. In the near future 27 research projects, partly ongoing and partly grant proposals, will implement microarray technology. On the longer term, microarray technology will be integrated and concentrated in the GenomeCenter Maastricht, which is currently being founded. In this Center microarray technology will be developed in conjunction to linkage facilities, mathematical genetics and molecular biology.

For microarray production and for data acquisition, the Genetic MicroSystems 417 Arrayer (GMS417) and the Genetic MicroSystems 418 Arrayscanner (GMS418) are available. The spotting mechanism of the GMS417 is a pin-and-ring system, using 4 spring-loaded 150mm pins. The GMS417 enables to produce 42 glass slides with identical, consistent patterns of DNA spots (basically in two spotting patterns) at moderate speed (~1 dot/sec). The solid pins combined with the relatively width rings allow the pick-up of an appropriate amount of fluid, prevents obstructions that might hamper the application of probe-DNA, and can efficiently be cleaned by the integrated washing and vacuum drying station. The GMS417 is a robust and open system that allows further development and modification of the hardware. Delicate points detected so far are the pins (length and straightness) and the springs that press the pins to the glass surface. Limiting factors at this moment are the software that does not provide the possibility to use 384-wells microtiter plates, the limited number of plates (n=3) that can be loaded at the same stage of a run, and the large amount of water used for rinsing the pin-and-ring system. So far, the GMS418, a two color confocal laser scanner system, could not be evaluated because of a major defect upon delivery.

Gene expression microarrays will be produced, using commercially available collections, or specific subsets from these collections (e.g. kidney-microarray, cardiovascular-microarray), or libraries generated/collected by investigators. Application of gene expression arrays will be in the research of the pathogenesis of inherited diseases1,2,3,4, developmental processes2, cardiovascular diseases3, mitochondrial diseases1,2, carcinogenesis2, gene-environment interactions4, and pharmacology3. Microarrays of spotted (modified) oligonucleotides will also be developed, both for mutation screening and analysis1,2, and for polymorphism studies1,2,3,4,5. Strategies that will be evaluated include allele-specific oligonucleotides (ASO), allele-specific primer extension (APEX), general hexamer catcher-probes combined with gene-specific primers and oligonucleotide ligation assays (OLA).

Dept. of Molecular Cell Biology & Genetics, Division of Genetics, Maastricht University, P.O. Box 1475, 6201 BL Maastricht, The Netherlands. Tel: +31-43-3875843/3881427, Fax: +31-43-3877877. E-mail: F.vanderLoop@molcelb.unimaas.nl,   Bert.Smeets@molcelb.unimaas.nl

| Top of page | ArrayNL homepage |