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.
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