Target Discovery and Validation Reviews and Protocols

124 Røsok and Sioud
represent each gene. The individual mRNAs are identified through the generation
of 17- to 20-base signature sequences.
1.2.3. DNA Microarray
DNA microarray methods provide simultaneously hybridization-based monitoring
of relative expression levels of genes on a genomic scale, from two
sources.
Important advances have led to the development of DNA microarray technology.
First, large-scale sequencing projects have made it possible to assemble
collections of DNA corresponding to large fractions of genes in many organisms.
Second, development of chip-printing technology has made it possible to
generate microscopic arrays of large sets of immobilized DNA sequences.
Third, advances in fluorescent labeling and detection of nucleic acids have
made the use of DNA microarray simpler and more accurate. The term
“microarray” is normally used for two independently developed technologies:
cDNA microarrays (52) and GeneChip arrays, developed by Affymetrix (53).
The basic principle of the techniques is that cDNA representing expressed
genes from two different sources (normally test and control preparation), and
labeled with two different fluorophores, is hybridized simultaneously in competition
to gene-specific probes immobilized at predetermined positions at high
density on two-dimensional solid microarrays. These solid supports, often
called chips, may contain several thousand sequences, and they represent the
total transcriptome in the investigated cell type. Capturing of the target molecules
by the probes on the array through sequence complementation enables
determination of the relative abundance in the two samples of mRNA corresponding
to each arrayed gene. Detection of the hybridization signal is performed
by a laser scanner that measures ratio of fluorescence intensities of the
two flours emitted by each gene spot. The two most common platforms for gene
expression analysis use in situ synthesis of oligonucleotide probes or robotic
deposition of cDNA probes to the solid support. For discussions about aspects
of the array production, sample labeling, hybridization, data analysis, and so on,
we refer the readers to comprehensive reviews (54–57).
Advantages with microscale assays are reduction of reagent consumption;
minimized reaction volumes, which increases sample concentration; and accelerated
reaction kinetics. This assay enables analyzing expression of all the
genes in the organism of interest in one reaction. Because each spot on the array
represent a known sequence, the identity of a gene is known once a signal is
detected. Microchips allow true parallelism and automation, which increases
speed of biochemical research.
Problems concerning limiting amounts of mRNA may be overcome by using
amplification methods that will not distort the representation of the various
mRNAs (2,3). Monitoring of relative expression levels is restricted to genes