Development of a Toxicological Gene Array and Quantitative Assessment of This Technology

Abstract

High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to ␤-naphthoflavone (␤-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after ␤-NF administration. Significant upregulation of Cyp1a2 was noted at ␤-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15-and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18 -60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability.

Application of toxicogenomic analysis to risk assessment of delayed long-term effects of multiple chemicals including endocrine disruptors in human fetuses

“…In the past 5 years, toxicogenomics has become a remarkable scientific field that combines studies of genetics, genomewide mRNA expression, cellwide and tissuewide protein expression, bioinformatics, and toxicology to understand the roles of gene-environment interactions in diseases (Bartosiewicz et al 2000;Iannaccone 2001;Lobenhofer et al 2001;Medlin 1999;Nuwaysir et al 1999;Rockett and Dix 1999;Tennant 2002). Toxicogenomic analysis using DNA microarray is a powerful and high-throughput method for monitoring the expression of thousands of genes simultaneously Duggan et al 1999;Hossain et al 2000;Komiyama et al 2002;Rockett and Dix 1999).…”

Analysis of the Heat Shock Response of Neisseria meningitidis with cDNA- and Oligonucleotide-Based DNA Microarrays

“…The production of cDNA microarrays requires the PCR amplification of all genes to be included in the array. In addition, PCR products can have disadvantages, such as varying degrees of GC content and possible inclusion of sequence stretches with high homology to alternative ORFs, which causes nonspecific signal to interfere with the specific target sequence (3,18). Regions sharing a homology above 75% of the length of oligonucleotide probes were recently shown to be sufficient for cross-hybridization (9).…”

How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

“…Fluorescence-inherent sources of error can be easier certified, however only, if the necessary controls and standards are included in the experiment and if the scanners are properly characterized. Fluorescence-based uncertainties are related to fluorophore labeling of the target [125,126] and to problems linked to measurements of fluorescence intensities, i.e. the sensitivity of the label's spectroscopic properties to chromophore microenvironment [127,128] and to dye-dye interactions [129] as well as to instrumentspecific effects.…”