Microarrays are a collection of DNA probes that are usually bound in defined positions to a solid surface, such as a glass slide, to which sample DNA fragments can be hybridised. The probes are generally oligonucleotides that are ‘ink-jet printed’ onto slides (Agilent) or synthesised in situ (Affymetrix). Labelled single-stranded DNA or antisense RNA fragments from a sample of interest are hybridised to the DNA microarray under high stringency conditions. The amount of hybridisation detected for a specific probe is proportional to the number of nucleic acid fragments in the sample.

One-colour or two-colour arrays?

A major design consideration in a microarray experiment is whether to measure the expression levels from each sample on separate microarrays (one-colour array) or to compare relative expression levels between a pair of samples on a single microarray (two-colour array) (Figure 2). The overall performance of one-colour and two-colour arrays is similar (2).

Figure 2 One-colour vs two-colour arrays.

In two colour microarrays, two biological samples (experimental/test sample and control/reference sample) are labelled with different fluorescent dyes, usually Cyanine 3 (Cy3) and Cyanine 5 (Cy5). Equal amounts of labelled cDNA are then simultaneously hybridised to the same microarray chip. After this competitive hybridisation, the fluorescence measurements are made separately for each dye and represent the abundance of each gene in one sample (test sample, Cy5) relative to the other one (control sample, Cy3). 

The hybridisation data are reported as a ratio of the Cy5/Cy3 fluorescent signals at each probe. By contrast, in one colour microarrays, each sample is labelled and hybridised to a separate microarray and we get an absolute value of fluorescence for each probe.

Limitations of microarrays

Hybridisation-based approaches are high throughput and relatively inexpensive, but have several limitations which include (6):

  • reliance upon existing knowledge about the genome sequence
  • high background levels owing to cross-hybridisation
  • limited dynamic range of detection owing to both background and saturation signals
  • comparing expression levels across different experiments is often difficult and can require complicated normalisation methods