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Introduction to two colour microarrays

The microarray menus provide facilities for the design and analysis of microarray experiments. Microarray experiments are used in genetics to screen RNA samples against libraries of DNA/RNA samples/fragments.

Two Channel Microarrays

A microarray experiment consists of a series of slides that have been printed with spots of DNA from a library. The DNA printed onto the slides or chips is known as the probes. The spots can be printed using a robot or ink jet printer to transfer DNA from the library (often held in 96 or 384 well plates) to the slide. Alternatively the probes can be laid down on the slide chemically, building up the probes base by base. RNA or DNA from two sources, known as the targets, (e.g. individuals, cell lines, cells at different times or under different conditions etc.) are then bound to 2 fluorescing dyes (Cy5 = red and Cy3 = green, note: to help remember the colour of the dyes Cy3 and Cy5, the words three and green share 3 letters). Two dyed targets are then added to a slide in equal amounts. The target RNA binds to the probe DNA on the slide, where there is a long sequence of complementary bases (i.e. the base A binds with T and C with G). This step is known as hybridization, and is normally carried out over a period of time under controlled conditions (e.g. temperature etc.) After the hybridization step, when the targets are left to bind to the probes, the unbound target DNA is washed from the slides. The amount of bound DNA for each target is read by scanning the slide, often with a confocal scanning laser which produces an image of the slide. The scanner uses two lasers tuned to the Cy3 and Cy5 wavelengths to produce a green and red image.

The following image shows a section of a microarray slide printed by one pin. The complete microarray slide can be seen here.

The two images are then input into an image analysis package (such as Spot, Genepix or ScanAnalyze) to produce a data file with a single level of intensity for each colour/channel and each spot. The image analysis routine should locate each spot, and then read an average value for the signal for each spot (the foreground value). In addition it may also provide background values for each spot to allow for background correction.

See the two channel microarray example to view how the menus can be used to analyse a two channel experiment.

The microarray menu has been designed so that you can progress easily through each step of an analysis. For example, the first section is for designing a two colour microarray experiment. This is followed by a section for importing data and then calculating log-ratios. Further sections are available for exploring the data and then normalizing. The data is explored and then normalized. The Analyse section can then be used to estimate the effects and perform empirical Bayes estimation to add power from the parallelism of the many probes. The False Discovery rate can then be accessed to aid in deciding how many probes to follow up. The final two sections can be used to graphically visualize the results and to cluster probes and targets to look at groups or for structure in the experiment.

Only the menus appropriate for two colour microarrays are listed below. The excluded menus are used for analyzing Affymetrix/single colour microarrays.


Generate Two Channel Design
Check Two Channel Design
Open Microarray Data Files
Density Plot
2D Plots
Spatial Plot
Two Channel
Estimates Two Channel Effects
Empirical Bayes Estimates
False Discovery Rate
Volcano Plot
Q-Q plot
Updated on April 29, 2019

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