Plant Biology Guide: Spotted Array

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.

Another common method is the single-extract technique (Causton et al., 2003) . This involves the use of commercial arrays such as the Affymetrix GeneChip TM (www.affymetrix.com), and relies upon the relationship of spot intensity measures of oligonucleotide probes hybridised to different array chips. This has the advantage that comparisons can be made between chips from multiple sources, allowing for changes in experimental focus and the sharing of datasets with other researchers. Following hybridisation the array is scanned. This produces a 16-bit greyscale TIFF image. The relative fluorescence of each spot is then ascertained from the image. Microarray image analysis software is used to measure pixel intensities. The intensity of each spot is normalised, allowing comparisons of spots within and between array(s). Normalisation itself is an adjustment of the average value of an experimental array to a baseline array.