The Degradation of dG Phosphoramidites in Solution.

The reaction of 2'-deoxynucleoside phosphoramidites with water is an important degradation reaction that limits the lifetimes of reagents used for chemical deoxyoligonucleotide synthesis. The hydrolysis of nucleoside phosphoramidites in solution has therefore been investigated. The degree of degradation depends not only on the presence of water but also on the specific nucleoside, 2'-deoxyguanosine (dG) being especially susceptible. Additionally, the nature of the group protecting the exocyclic amine on the nucleoside base strongly influences the rate of hydrolysis. For dG, the degradation is second order in phosphoramidite concentration, indicating autocatalysis of the hydrolysis reaction. Comparison of the degradation rates of dG phosphoramidites with different protecting groups as well as with phosphoramidites containing bases that are structurally similar to dG affords clues to the nature of how dG catalyzes its own destruction and indicates a direct correlation between ease of protecting group removal and propensity to undergo autocatalytic degradation.

Using RNA sample titrations to assess microarray platform performance and normalization techniques.

We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

The Agilent in situ-synthesized microarray platform.

Microarray technology has become a standard tool in many laboratories. Agilent Technologies manufactures a variety of catalog and custom long-oligonucleotide (60-mer) microarrays that can be used in multiple two-color microarray applications. Optimized methods and techniques have been developed for two such applications: gene expression profiling and comparative genomic hybridization. Methods for a third technique, location analysis, are evolving rapidly. This chapter outlines current best methods for using Agilent microarrays, provides detailed instructions for the most recently developed techniques, and discusses solutions to common problems encountered with two-color microarrays.

Rapid quantitative profiling of complex microbial populations.

Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities--a 'census'--is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10,462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples--the current 'gold standard' method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.