IMS Microarray Core facility (IMCF)- Technology
Affymetrix GeneChip™ Instrument System
The Affymetrix GeneChip™ Instrument System comprises a hybridisation oven, fluidics station, confocal laser scanner and workstation. The basic principle involves hybridisation of your sample of interest ('target') to commercially made GeneChip™ arrays ('probe'). The target is usually total RNA (Gene expression arrays; total RNA is reverse transcribed to cDNA and labelled with biotin-UTP and biotin-CTP using in vitro transcription of cDNA to cRNA) or genomic DNA (DNA analysis arrays) isolated from your sample of interest (see protocols for full details). Following hybridisation of the labelled-target to the probe in the hybridisation oven, the array is processed in the fluidics station (includes wash steps, incubation with the phycoerythrin-streptavidin substrate and a fluorochrome amplification step) using a protocol optimised for the specific GeneChip™ used. The array is then imaged on a confocal laser scanner which detects the phycoerythrin fluorochrome linked to the biotin-label on the bound target. Microarray Analysis Suite software controls the fluidics station and scanner, thereby limiting inter-assay variability, and provides the user with normalised expression data for paired samples. The software automatically calculates data from all 11-20 oligonucleotide probe pairs for a target (see below for further details) and integrates the data to allow expression analysis, nucleotide analysis, SNP detection or allele detection from a specific GeneChip™ array.
Affymetrix GeneChip™ Arrays
Affymetrix GeneChip™ expression arrays enable the analysis of tens of thousands of mRNA transcript levels from a single sample. The GeneChip™ arrays are designed to maximise sensitivity, specificity and reproducibility. There is no need for preparation of thousands of probes with the associated time and high costs involved. Commercial manufacture of a GeneChip™ array, requires synthesise of hundreds of thousands of unique oligonucleotides in situ in precise locations on a small glass slide using photolithography and solid phase chemistry. This unique state-of-the-art design strategy results in highly specific, sensitive, reproducible data. Sequence information from databases such as UniGene and Genbank is used to select and design the synthetic oligonucleotides, with each 'target' on a GeneChip™ expression array represented by an average of 11-20 pairs of 25-mer oligonucleotide probes. Each probe pair consists of one perfect match oligonucleotide and one mismatch oligonucleotide (a single base mismatch at nt 13). This unique pairing strategy helps identify and subtract non-specific hybridisation and background signal (see technical note). The combined signal from all probe pairs for a specific 'target', is used to determine the signal intensity for that 'target' in your sample.
Each array also contains multiple house-keeping gene probes and other reference gene probes, allowing for normalisation and quantitation of data from multiple experiments. In addition, 'spiking' of target material allows the efficiency of labelling and hybridisation to be monitored using these controls. For example, the human annotated GeneChip™ array (HG-U133A) contains almost 500 000 spots corresponding to oligonucleotide probe sets for approximately 22000 transcript variants, representing 16000 fully annotated unique human genes plus reference genes. Each GeneChip™ array is provided in a sealed unit to limit background problems caused by dust.
A broad range of GeneChip™ arrays are available and they can be used not only for expression analysis, but also for genotyping, resequencing and disease management assays. These include expression arrays for human, mouse, rat, E.coli, S.cerevisiae, Arabidopsis, Drosophila, C. elegans and P. aeruginosa, as well as Human Mapping 10K and Mapping 100K SNP arrays, p53, SARS and Custom resequencing DNA analysis arrays. Custom-made arrays can also be analysed on this system, including custom made GeneChip™ expression arrays (low or high density- but very expensive, have to buy whole batch so is usually only a feasible option for a consortium) and GenFlex Tag arrays which have the potential to allow investigation of protein-protein interactions as well as transcript expression analysis.
An overview of Affymetrix GeneChip™ technology is available from the technology tab within the Affymetrix website.
A diverse range of scientific publications utilising Affymetrix GeneChip technology is available in the literature.