Principle of cDNA microarray assay of gene expression
A microarray is a set of short Expressed Sequence Tags (ESTs) made from a cDNA library of a set of known (or partially known) gene loci. The ESTs are spotted onto a cover-slip-sized glass plate, shown here as a 8x12 array. In practice, microarrays of many thousand ESTs are possible.
A complete set of mRNA
transcriptome) is prepared from the tissue of an experimental treatment or
condition, e.g. fish
fed a high-protein diet, or an individual with breast cancer.
Complementary DNA (cDNA) reverse transcripts are
prepared and labelled with a [red] fluorescent dye. A control library is
constructed from an untreated source, e.g.
a standard fish
diet, or non-cancerous breast tissue; this library is labelled
with a different fluorescent [green] dye. The experimental and control
libraries are hybridized to the microarray. A Dual-Channel Laser excites
the corresponding dye, and the fluorescence intensity
indicates the degree of hybridization that has occurred.
Relative gene expression is measured as the ratio of the two
fluorescence wavelengths. Increased expression or “up-regulation” of genes in the
experimental transcriptome relative to the control will be
visualized as a "hotter" red “pseudo-colour,” and decreased expression or
shows as a "cooler" green. Intensity
of color is proportional to the expression differential. Unchanged, constitutive expression (1:1 ratio of experimental
to control) shows as a neutral black.
In the following example, cDNA microarrays are
used to measure life-stage
tissue specific patterns of gene expression.