A Practical Approach, Second Edition=Ronald D. Ho.pdf

FUNCTIONAL GENOMICS AND PROTEOMICS 623the data should be confirmed and validated by additional approaches, such as real time-PCR orNorthern blotting.With these tools now readily available to the scientific community, gene expression profilesthat are characteristic of modifications caused by developmental or reproductive toxicants can andshould be identified. Experimental designs presently adhere to traditional teratogen testing methods,i.e., comparing patterns of gene expression in experimental samples vs. those observed in controls.This section specifically reviews two recent methods for global gene expression profiling, namelymicroarrays and SAGE, that may be used in toxicology studies. With these tools, global changesin gene expression profiles can be identified and characterized. These are critical first steps forunderstanding the mechanisms of action of reproductive or developmental toxicants or for predictivepurposes in the identification of potential teratogens.B. MicroarraysThe platform typically associated with gene expression profiling is the microarray, also known asthe “DNA chip” or “gene chip.” 5 Microarrays come on different platforms and can be custommanufactured in-house or purchased from selected biotechnology companies. The common themeconcerning microarrays is that scientists are able to study the expression of a large number of genessimultaneously in a single experiment. Experiments that in the past would have taken several yearsto complete can now be performed within a matter of days. Aside from the high throughput natureof the microarray technology, one of the most prominent features is the ability to study novel andpreviously uncharacterized genes. Thereby previously unknown genes or genes never before linkedto the experimental questions being pursued may be implicated. This, in turn, provides opportunitiesto target these newly implicated genes as part of drug discovery processes or to study their function,regulation, and cellular networks. In toxicological studies, discovery of novel genes that are perturbedin response to toxicological stimuli may enhance understanding of the underlying mechanismsof toxicity associated with a specific compound.In retrospect, microarray methodology is merely a recent extension of well-established methodsto assess gene expression by hybridization. Such methods have been successfully applied for manyyears. The first hybridization-based method, Southern blotting, 6 was used to study only a limitedset of genes. Subsequent methodologies such as “dot-blots,” enabled investigators to explore alarger set of genes that are deposited on porous nylon membranes. 7 What distinguishes microarraysfrom the aforementioned methods is primarily the use of glass as the solid support. Contrary tonylon membranes, glass (being a nonporous material) enables high-density and spatially reproducibledeposition of target DNA, resulting in the ability to screen thousands of genes simultaneously.Using a nonporous support has also simplified hybridization kinetics and improved image acquisitionand processing procedures. 8 Concurrent to developments in fluorescent labeling of nucleicacids, microarrays enable scientists to evaluate gene expression from both experimental and controlsamples on the same slide simultaneously. There are two major types of DNA arrays: complementaryDNA arrays and oligonucleotide arrays. Several features, such as the length of target DNA,the method for DNA deposition, and the slide chemistry, distinguish the two types of arrays.1. Complementary DNA (cDNA) ArraysComplementary DNA arrays, better known as cDNA arrays, were initially developed in the laboratoryof Patrick Brown and colleagues from Stanford University. Their first publication describingthe use of this technology with a small subset of Arabidopsis genes appeared in 1995. 9 Since then,cDNA arrays have become the most popular microarray platform, and it is now quite common forinstitutions and departments to have their own core microarray manufacturing facilities. The mainreasons for the popularity of cDNA arrays are:© 2006 by Taylor & Francis Group, LLC