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.

An analytic solution of the Förster energy transfer problem in two dimensions is presented for the case in which the orientation factor is independent of the donor-acceptor distance, and both the donors and acceptors are randomly distributed in a plane. A general solution based on the method of Förster is possible since all distances are measured in units of R0. The analytic solution is extended to the cases of donors embedded in structures that exclude acceptors, and donors that bind acceptors. The validity of the analytic solutions is demonstrated by comparison with numerical simulation calculations. Numerical approximations to the exact solutions are given for ease of computation. Specific applications to the case of fluorescence quenching of a membrane-bound donor by membrane-bound acceptors are presented.

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.

The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl 1-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process.Some bacteria of the genera Pseudomonas, Erwinia, and Xanthomonas can nucleate the crystallization of ice from supercooled water (1-3). Genes encoding the ice-nucleation active (Ina') phenotype have been cloned from Erwinia herbicola, Pseudomonas syringae, and Pseudomonas fluorescens (4-7). Theoretical considerations (8), as well as the actual sequence of the inaZ gene from P. syringae (6), suggest that the bacteria synthesize a template for ice-crystal formation, rather than an enzyme. The translation product predicted from the inaZ sequence is a protein with repetitive primary structure; its tertiary structure, which might also be repetitive, could provide considerable insight into the mechanism of ice nucleation. The ice nuclei of P. syringae are associated with its outer membrane (9) and are believed to contain both protein (9-11) and lipid (11) components. One report has suggested that the inaZ product is a phosphatidylinositol synthase, and that the lipid phosphatidylinositol is a key component of the water-binding template (12).Here, we argue that the active component of bacterial ice nuclei is a protein, the product of inaZ. We have reached this conclusion after purifying the InaZ protein. To Ice-Nucleation Spectra. Ice-nucleation frequencies were measured by a drop-freezing method with an instrument constructed as described by Vali (16). For analysis of column fractions, 20 drops of 10 ,u1 per dilution were examined at a dilution interval of 10-2; in all other cases, 40 drops of 10 p.1 per dilution were tested, with a dilution interval of 10-1. All spectra of subcellular fractions were normalized to the frequency per cell, by dividing the frequency per ml value for a given sample by the ratio (volume of sample)/(number of cells used to prepare sample).Construction of pMWS10. The region of DNA encoding the P. syringae S203 inaZ gene (6) was digested with restriction enzymes Aha III (cutting at nucleotide 775) and EcoRI (cutting at nucleotide 4453), resulting in a fragment beginning 23 base pairs 5' of the initiator codon. The EcoRI end was converted to a HindIII end by addition of a linker, and the fragment was inserted into pKK223.3 (17), so that inaZ was placed downstream of the tac promoter. The construct retained the original ribosome binding site of inaZ. The plasmid pMWS10 ...

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

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.

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The time-resolved fluorescence intensity and anisotropy decays of cis- and trans-parinaric acids and phosphatidylcholines labeled with trans-parinaric acid have been characterized in bilayers formed by several phosphatidylcholines and by dipalmitoylphosphatidylcholine-cholesterol mixtures, at several temperatures. Both a conventional free-running nitrogen flashlamp and the novel synchrotron source at the Stanford Linear Accelerator Center (SLAC) were used as excitation sources for a modified single photon counting fluorescence lifetime apparatus. The measured emission decay kinetics of both isomers of parinaric acid were biexponential in all but one of the lipid systems examined. The fluorescence anisotropy of parinaric acid was large and constant in gel phase lipids, but showed a very rapid (approximately 2 GHz) decay of large amplitude in fluid lipids. In all lipid systems studied, the fluorescence anisotropy decayed to a nonzero asymptote, in striking contrast to the behavior observed in viscous solvent solutions. The asymptotic anisotropy was used to calculate an "order parameter" of the emission transition dipole. The value of the order parameter is quite close to that obtained by deuterium NMR. Cholesterol increased the order parameter measured in fluid dipalmitoylphosphatidylcholine but did not substantially affect the rate of angular relaxation. Experiments conducted with trans-parinaroylphosphatidylcholines yielded results virtually identical with those obtained with trans-parinaric acid.