Microarrays and sequencers: Expressions in array technology

Microarray technology is now a major tool available to the geneticist. The technology allows researchers to look at the expression of a vast array of genes simultaneously, and myriad applications have been found in the last few years. But deciding on the best microarray system for the needs of the research is a difficult task, with several choices available.

Microarray technology requires several components. First the microarray has to be made, and several different technology platforms exist to accomplish this task. The cheapest option is a quill pin system, which uses pins to print, or spot the DNA or oligonucleotide onto a microscope slide or membrane.

"The Australian market is pretty saturated with the VirTek instruments [now made by BioRad]," says Dr John Barlow, of the Australian Genome Research Facility (AGRF).

The AGRF fabricates arrays for Australian researchers to use in their gene expression studies, with human and mouse gene arrays available off-the-shelf, as well as custom arrays. Other microarray manufacturing facilities are also available at various institutes around Australia.

"We can print approximately 30,000 elements, or spots, onto a standard microscope slide. You can make 120 slides per run, and it takes about three days from start to finish," says Sean Grimmond, head of the Microarray Facility at the Institute of Molecular Bioscience at the University of Queensland. His facility uses both amplified cDNAs and long oligonucleotides to custom-make arrays covering human and mouse genomes.

Andrew Holloway, from the Peter MacCallum Cancer Institute, also uses the VirTek system. The institute has several standard arrays, including 10.5K (10,500 genes) human and 15K mouse cDNA arrays, and will be adding a 19K human oligo array shortly. These are primarily used for expression profiling, particularly for tumours, he says. "People will continue to do expression profiling forever," Holloway believes. "It's really expression profiling that is driving the array field at the moment."

But the disadvantages of microarrays manufactured using the quill pin method include variability between slides and spots, resulting in less reproducible results. In addition, the method is prone to contamination as the pins directly contact the sample and the slide. "At the moment we're using printing technology, but a lot of people are looking towards non-contact printing -- piezoelectric or inkjet printers," Grimmond says.

This technology has the advantage of being more automated and promises to give higher quality arrays, but is not yet widely available in Australia. Perkin Elmer is one manufacturer of these instruments.

At the pinnacle of current chip manufacturing technology are photolithographic processes, which build the oligonucleotide directly on the chip. These are far more expensive than the pin systems. Affymetrix is currently the only company offering photolithographic microarrays, although there are a number of other companies developing similar technology including German company Febit and US-based NimbleGen Systems.

Affymetrix's GeneChip is a sealed unit, which is hybridised with the sample under very controlled conditions, according to Bren Collinson, who is the managing director of Affymetrix's Australian distributor Millennium Science. Currently, their human array comprises two chips, with a total of 40,000 genes representing essentially the entire human genome. "Sometime in the future, it will have the whole genome on one chip. They are moving in that direction for all of the model organisms," says Collinson.

Robert Henke, a product specialist for Millennium, agrees. He says the number of genes per chip is increasing, while the area taken up by each spot has been reduced and the length of the oligo has also decreased. In addition to the human GeneChip set, Affymetrix has genomic chips for a number of model organisms including E. coli, yeast, C. elegans, Drosophila, Arabidopsis, mouse and rat. Custom-made chips are also available, but knowledge of the sequence is required.

At present there are six facilities in Australia using the Affymetrix GeneChip system, which requires specialised equipment for hybridisation and scanning. Among these are the Walter and Eliza Hall Institute, in Melbourne, and the Garvan Institute of Medical Research in Sydney.

"It works out to be more expensive than using glass slide microarrays, but the quality is better. Though it costs more, you don't have to run as many," says Melinda Frost, a research associate at the Garvan Institute involved in running their GeneChip system. Once the chip has been fabricated, and the hybridisation performed, a scanner is used to analyse it. Again, there are quite a few brands available, including the GenePix series manufactured by Australian company Axon Instruments.

While scanner systems have software for the analysis of microarrays, there are other informatics packages available, some in the public domain. Bioinformatics is a crucial part of microarray research, as the amount of data generated can be considerable, and the choice of analysis tools really depends on the complexity of the research. "Image analysis allows you to work out which genes have gone up and which have gone down, but then if you want to look at changes across a series of arrays, then you need a statistics package," says Grimmond.

According to Grimmond, one of the more popular software packages is Biodiscovery's Imagene, which is used by a lot of Australian researchers. Another program, Digital Genome, is more commonly used by US and Japanese researchers. Other commercial programs used include OmniViz and Spotfire. "It's now moving from lower-end packages to high-level bioinformatics," notes Collinson.

Making it new

While expression profiling remains very popular, new uses for microarrays have appeared. Affymetrix, for example, has developed several new uses including array-based resequencing and SNP (single nucleotide polymorphism) analysis. The company's first SNP GeneChip provides 1500 SNPs for analysis, but in the near future, the company plans to release a 10,000 SNP array. Robert Henke, a product specialist for Affymetrix distributor Millennium Science, believes that ultimately Affymetrix will put 100,000 SNPs on one chip.

"It's still early days, but it will be a significant product," he says. Bren Collinson, managing director of Millennium Science, says that Affymetrix is pushing its product platform into the health management arena, using arrays as a diagnostic tool. It's an important area, he says, but not quite there yet.

Deon Venter, from the Murdoch Children's Research Institute, is also keen on using microarrays as a diagnostic tool. At a research level Venter and other scientists are already using arrays to characterise and profile tumours. "One of my drives is to use the technology to diagnose and profile cancers, using array systems as part of a clinical diagnostic system. You can do it, but it's still too high a cost for clinical pathology labs," he explains.

Venter believes that the next interface will be pharmacogenomics, which would combine information about a patient's condition, for example their tumour profile, with knowledge of their individual genetic makeup to choose tailor the most suitable drug regime. This would allow doctors to avoid drugs with potentially severe side effects in a given patient, in addition to choosing the most effective drug against the tumour.

"It makes enormous sense to interface pharmacogenomics with array genetics to look at the potential for interactions," he says.

Another application of array technology examines living cells in an array format, by transfecting cDNA constructs into cells grown on slides. The expression of the DNA in the cell can then be examined, even localised to a particular subcellular compartment, says Sean Grimmond, from the Institute of Molecular Bioscience.