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Counteracting dye bias effects when using two-colour arrays

One issue for two-colour arrays is related to dye bias effects introduced by the slightly different chemistry of the two dyes. It is important to control for this dye bias in the design of your experiment, for example by using a dye swap design or a reference design (Figure 3). In a dye swap design, the same pairs of samples (test and control) are compared twice with the dye assignment reversed in the second hybridisation. However, the most common design for two colour microarrays is the reference design in which each experimental sample is hybridised against a common reference sample. 

Figure 3 A dye swap design involves hybridising the same two samples (test and control) twice. In one hybridisation, the control sample is labelled with Cy3 dye (cyan) and the test sample with Cy5 dye (red). In the second hybridisation dye assignment is reversed. In a reference design, both test and control samples are labelled with one dye (usually Cy5), while a reference sample is labelled with the other dye (usually Cy3) and co-hybridised.

Performing replicates

Replicates are essential for reliably detecting differentially expressed genes in microarray experiments. Without replicates, no statistical analysis of the significance and reliability of the observed changes is possible; the typical result is an increased number of both false-positive and false-negative errors in detecting differentially expressed genes. There are two types of replicates: technical replicates and biological replicates (see the section on Number of replicates later in the course).

In Expression Atlas, a minimum acceptable number of biological sample replicates (three) is enforced to ensure sufficient statistical power to detect differential expression (3).