Overview Of Gene Expression Analyses

DNA microarray technology was developed in the early 1990s. Its refinements, together with great improvements during these years, defined the technology as the platform of choice for the application of gene expression profiling in many fields, from toxicology and ecotoxicology (11), to drug development and diagnostic tools (8).

Recently, the U.S. Food and Drug Administration approved the "Amplichip," the first DNA microrarray application in clinic (12) to help physicians to test the dosage of drugs that are differently metabolized by cytochrome P450 enzyme variants.

The field of DNA microarray has evolved from the key insight of Ed Southern (13), who showed that it is possible to attach nucleic acid to solid support. The resulting Southern blot can be viewed has the first DNA array (14). It was only a small step to improve the technique to filter-based screening of clone libraries, which introduced a one-to-one correspondence between the clone and the hybridization signal (15) in a fixed position; in this way the clone could be uniquely identified and information about it accumulated.

The subsequent explosion of array technologies has been sparked by two key innovations. The first is the use of non-porous solid support, such as glass, which has facilitated the miniaturization of the array and the development of fluorescence-hybridization detection (16, 17, 18). The second critical innovation has been the development of methods for high-density spatial synthesis of oligonucleotides, which allows the analysis of thousands of genes at the same time. Because DNA cannot bind directly to the glass, the surface is first treated with silane to covalently attach reactive amine, aldehyde, or epoxies groups that allow stable attachment of DNA, proteins, and other molecules.

The nucleic acid microarrays use short oligonucleotides (15-25 nt), long oligonucleotides (50-120 nt), and PCR-amplified cDNAs (100-3,000 bp) as array elements. The short oligonucleotides are primarily used for the detection of single nucleotide polymorphisms (SNPs). Indeed the destabilization, caused by mispairing of only one mismatch, is maximized by the short oligonucleotide (18). The long nucleotides and the PCR-amplified cDNAs produce strong signals and high specificity (19), unambiguous sample identification, and affordability.

The cDNA elements are readily obtained from cDNA libraries. They are typically used when only a limited part of genome is known. With this technology cells or tissues are exposed to toxicants, drugs, and then gene expression is measured by collecting mRNA, converting mRNA to labeled cDNA, hybridizing it to the DNA array, staining it with an appropriate dye, and visualizing the hybridized genes using a fluorom-eter (16,19,20) (see Fig. 1). The raw data are analyzed using bioinformatics software and databases. The aim is to obtain meaningful biological information such as patterns of relative induction/repression levels of gene expression, participation in biochemical pathways, and (in the most favorable cases) "genetic signatures." Several exhaustive reviews are available both on the practical aspects of DNA microarrays and the analysis of data (21,22,23,24).

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