|
|
Microarrays and gene expression analysis
8/1/03. By Richard Twyman
Microarrays can be used to analyse the expression of tens of thousands of genes simultaneously.
|
Microarrays are miniature devices containing thousands of DNA sequences stuck on at different positions (addresses). Hybridisation to complex mixtures of labelled DNA molecules, prepared from cellular RNA, shows the relative expression levels of thousands of genes. This can be used to compare gene expression levels within a sample or look at differences in the expression of
specific genes across different samples.
Key principles
-
RNA isolated from a particular cell type or tissue comprises a complex mixture of different RNA transcripts. The abundances of individual transcripts in the mixture reflect the expression levels of the corresponding genes.
-
A microarray is a small device, about the size of a microscope slide, with thousands of different known DNA sequences immobilised at different addresses on the surface.
-
Each of these DNA sequences can participate in a hybridisation reaction.
-
If a complex DNA mixture copied from the above RNA is labelled and hybridised to the microarray, the strength of the signal at each address shows the relative expression levels of the corresponding gene.
Microarrays can be used to compare gene expression levels within a sample or look at differences in the expression of specific genes across different samples.
How does it work?
Single DNA strands with complementary sequences can pair with each other and form double-stranded molecules. This hybridisation process occurs with such specificity that a labelled DNA probe - a single DNA strand carrying a label that allows it to be detected — can pick out a matching partner, the target, in a complex mixture containing millions of different sequences.
Microarrays apply the hybridisation principle in a highly parallel format. Instead of one target, thousands of different potential targets are arrayed on a miniature solid support. Instead of a unique labelled DNA probe, a complex mixture of labelled DNA molecules is used, prepared from the RNA of a particular cell type or tissue.
The abundances of individual labelled DNA molecules in this complex probe reflect the expression levels of the corresponding genes. When hybridised to the array, abundant sequences will generate strong signals and rare sequences will generate weak signals.
The strength of the signal thus represents the level of gene expression in the original sample.
How is it used?
Expression analysis with microarrays can be used to determine what genes are expressed in a particular cell type or tissue and to compare the expression levels of different genes.
It can also be used to compare gene expression across different but related samples, such as disease vs healthy tissue. A gene expressed only in the disease sample, for example, might represent a useful drug target. Comparative expression analysis can be achieved by comparing duplicate microarrays hybridised to complex probes prepared from the alternative samples.
A refinement of the technique, in which RNA from the related samples is labelled with different fluorescent molecules, allows this analysis to be carried out on a single microarray.
