WATCH DNA Microarray Analysis of Complex Biologic Processes

DNA microarrays, or gene chips, allow surveys of gene expression, ( i.e., mRNA expression) in a highly parallel and comprehensive manner. The pattern of gene expression produced, known as the expression profile, depicts the subset of gene transcripts expressed in a cell or tissue. At its most fundamental level, the expression profile can address qualitatively which genes are expressed in disease states. However, with the aid of bioinformatics tools such as cluster analysis, self-organizing maps, and principle component analysis, more sophisticated questions can be answered. Microarrays can be used to characterize the functions of novel genes, identify ge es in a biologic pathway, analyze genetic variation, and id ntify therapeutic drug targets. Moreover, the expression profile can be used as a tissue or disease “fingerprint. ” This review details the fabrication of arrays, data management tools, and applications of microarrays to the field of renal research and the future of clinical practice. Recent advances in functional genomics have made possible new approaches to the diagnosis and management of a wide array of renal disorders. With new tools to explore gene expression and regulation, researchers can understand better the molecular basis of disease. Of the estimated 30,000 human genes, only 30% are functionally understood. Moreover, fewer than 3% of identified genes have been characterized in renal disease, underscoring the need for functional genomics techniques in renal research. Although recent research has uncovered the genetic basis of hereditary disorders, including familial focal segmental glomerulosclerosis and adult polycystic kidney disease, using traditional genetic mapping techniques, more comprehensive approaches are needed to identify the complex sequence of perturbations that underlie polygenic conditions such as hypertension and chronic renal insufficiency (1–3). Among the most powerful of these new tools are DNA microarrays, capable of genome-wide profiles of mRNA expression. A DNA microarray, or gene chip, is a matrix of thousands of cDNA or oligonucleotides imprinted on a solid support (4,5). Labeled mRNA from the tissue of interest is hybridized to its sequence complement on the array to provide a measure of mRNA abundance in the sample. The hallmark of the microarray experiment is the expression profile, the pattern of gene expression produced by the experimental sample (Figure 1). Arrays composed of DNA fragments are not new (6,7). Howev r, early arrays included only a small set of genes thought to be involved in the process being studied. Significant improvements in substrate materials, robotics, and signal detection have made possible miniaturization of arrays with the result that hundreds of thousands of oligonucleotides can be arrayed on a square-centimeter chip. This important feature makes it possible to study gene expression without specifying in advance which genes are to be studied. Thus, DNA microarrays permit systematic and comprehensive surveys of gene expression in an efficient manner. Principles of Microarray Technology Microarray Structure Two variations of microarrays exist: ( 1) customized cDNA microarrays composed of cDNA or oligonucleotides and ( 2) commercially produced high-density arrays, e.g., Affymetrix GeneChip (Affymetrix, Santa Clara, CA), containing synthesized oligonucleotides (8). The first type of array can analyze RNA from two different samples on a single chip but requires a source of genes to be spotted onto the chip, usually expressed sequence tag clones or oligonucleotides. High-density commercial arrays provide expression analysis over a larger number of genes (12,000 human genes in the case of the Affymetrix GeneChip) but can analyze only a single sample on one chip and at considerable cost, making them unsuitable for largescale experiments in most academic laboratories. Both types of arrays produce sensitive and accurate expression data. Creating a cDNA Microarray Customized cDNA microarrays are fabricated by first selecting the genes to be printed on the array from public databases/repositories or institutional sources. High throughput DNA preparation, usually done by robotics systems, consists of Correspondence to Dr. Steven R. Gullans, Harvard Institutes of Medicine, 5th Floor, 77 Avenue Louis Pasteur, Boston, MA 02115. Phone: 617-525-5712; Fax: 617-525-5711; E-mail: sgullans@rics.bwh. harvard.edu