Microarray experimental difficulties
To minimise problems associated with microarray experiments it is imperative to carefully design the experiments. Variations in the data obtained is usually caused in three ways: measurement error associated with the reading of fluorescent signals; natural variability in the biological system, and technical variations due to the extraction, labelling and hybridisation of samples (Churchill, 2002) . It is therefore important to reduce the number of non-essential variables. Unless environmental factors such as light, temperature, humidity and time of sampling are being measured, they should be kept as constant as possible. The pooling of samples is useful in eliminating variability between individual samples (Bakay et al., 2002) . Replication of microarray experiments increases the confidence value of the results. Due to the high costs involved it is often not feasible to repeat experiments, especially when dealing with commercial arrays (Causton et al., 2003).
Types of array
Two major types of array platforms are currently in use: cDNA based and oligonucleotide based. Both types of array can be spotted on either porous substrates such as nylon or nitrocellulose, or non-porous surfaces such as a polymer or glass slide (Causton et al., 2003) . cDNA arrays are typically made up of PCR products spotted upon the array. This type of array enables clone banks and DNA from limited templates to be spotted. A disadvantage of DNA arrays is that contamination can occur between spots, and that it can be difficult to distinguish between results of closely related genes. The size of a PCR amplified fragment used on a cDNA array is in the order of 400bp to 1000bp (Causton et al., 2003) .
Oligonucleotide arrays
The second array class, oligonucleotides, are much smaller in size, varying between 25 and 80bp in length. These are either pre-synthesised and spotted onto the array, or synthesised directly on the array substrate. Oligonucleotide based arrays have advantages over cDNA arrays as they suffer less from contamination between spots, it is easier to standardise the sequence of each spot, and they can be purchased in a form that is ready for spotting.
Spotted microarray
The spotted microarray is hybridised with probes derived from the mRNA of the biological samples being assessed. In the technique known as dye swapping, in which multiple extracts are hybridised to arrays, the mRNA is typically reverse transcribed into cDNA and labelled with a spectrally distinguishable red (Cy5) or green (Cy3) fluorescent dye (Kerr and Churchill, 2001) . Samples are then washed over the microarray, allowing labelled cDNA strands complementary to sequences on the microarray to bind. Generally two dyes are used; if only one dye is used there is little measure of the amount of DNA targeted to any particular spot. However, the relative fluorescence of two dyes to each other can be measured, with the sample containing higher levels of transcript producing a greater signal.
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