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Results for “Oligonucleotide Array Sequence Analysis”

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PubMed2006

Discrimination and scoring using small sets of genes for two-sample microarray data.

Comparison of gene expression for two groups of individuals form an important subclass of microarray experiments. We study multivariate procedures, in particular use of Hotelling's T2 for discrimination between the groups with a special emphasis on methods based on few genes only. We apply the methods to data from an experiment with a group of atopic dermatitis patients compared with a control group. We also compare our methodology to other recently proposed methods on publicly available datasets. It is found that (i) use of several genes gives a much improved discrimination of the groups as compared to one gene only, (ii) the genes that play the most important role in the multivariate analysis are not necessarily those that rank first in univariate comparisons of the groups, (iii) Linear Discriminant Analysis carried out with sets of 2-5 genes selected according to their Hotelling T2 give results comparable to state-of-the-art methods using many more genes, a feature of our method which might be crucial in clinical applications. Finding groups of genes that together give optimal multivariate discrimination (given the size of the group) can identify crucial pathways and networks of genes responsible for a disease. The computer code that we developed to make computations is available as an R package.

Algorithms↗
PubMed2005

Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis.

An oligonucleotide microarray system has been specifically designed to detect and differentiate Bacillus anthracis from other bacterial species present in clinical samples. The pilot-scale microarray initially incorporated probes to detect six common species of bacteria, which were fully evaluated. The microarray comprised long oligonucleotides (50--70-mer) designed to hybridise with the variable regions of the 16S rRNA genes. Probes which hybridised to virulence genes were also incorporated; for B. anthracis, these initially included the pag, lef, cap and vrrA (for partial genotyping) genes. Hybridisation conditions were initially optimised to be run using 5 x SSC, 0.1% SDS, 50 degrees C for 16 h. The detection limits of the microarray were determined under these conditions by titration of chromosomal DNA and unlabelled amplicons followed by hybridisation to determine the levels of sensitivity that could be obtained with the microarray. Two different amplification methodologies were also compared-specific-primer based PCR and random PCR (with the labelling stage incorporated). Higher sensitivity was obtained using specific PCR primers, however, since one of the desired outcomes of a microarray-based detection system was the high discrimination that it offered, random amplification and labelling was used as the amplification method of choice. The length of hybridisation was investigated using a time-course, and 1--2h was found to give optimal and higher signals than 16 h incubation. These results indicate that microarray technology can be employed in a diagnostic environment and moreover, results may be obtained in a similar time-scale to a standard PCR reaction, but with the advantage that no a priori knowledge of the infectious agent is required for detection.

Bacillus anthracis↗
PubMed2006

Using PCR amplification to increase the confidence level of Salmonella typhimurium DNA microarray chip hybridization.

In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.

Base Sequence↗
PubMed2005

Comparative phylogenomics of pathogenic bacteria by microarray analysis.

DNA microarrays represent a powerful technology that enables whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. Such studies have dramatically increased our ability to differentiate between bacteria, highlighting previously undetected genetic differences and population structures and providing new insight into virulence and evolution of bacterial pathogens. Recent results from such studies have generated insights into the evolution of bacterial pathogens, the levels of diversity and plasticity in the genome of a species, as well as the differences in virulence amongst pathogenic bacteria.

Bacteria↗
PubMed2005

Fabrication and optimization of the multiplex PCR-based oligonucleotide microarray for detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum.

To detect and identify the pathogens responsible for sexually transmitted diseases (STDs) at the early stage of infection and with a high throughput, a new microarray with a bifunctional probe modification was prepared using Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum as a model system. During the fabrication of the microarray, an asymmetric fluorescently labeled multiplex PCR was introduced. The fabrication optimization proved that the best hybridization results would be obtained by spotting N. gonorrhoeae probe at a position near the side of the fluorescently labeled reverse primer within its target gene and spotting each probe at a concentration of 50 microM onto the aldehyde-derived glass slides using spotting solution S1 and using hybridization solution H2 for hybridization. The probes designed by our laboratory could specifically discriminate the pathogens of N. gonorrhoeae, C. trachomatis and U. urealyticum in the presence of the internal control on the microarray simultaneously and separately. By incorporating the key features of DNA microarray with those of multiplex PCR, the microarray provides a fast high throughput platform for multiple infections and multiple samples to be detected and identified simultaneously for STD clinics. It also provides a new platform for other diseases and gene mutations to be detected and identified at a high throughput.

Chlamydia trachomatis↗
PubMed2005

Analysis of covariance patterns in gene expression data and FT-IR spectra.

The aim of this study was to detect and interpret correlation patterns in several large data matrices from the same biological system using Partial Least Squares Regression (PLSR) in order to get information on the system under investigation. To do this, DNA microarray data and Fourier Transform Infrared (FT-IR) spectra from a designed study where Campylobacter jejuni was exposed to environmental stress conditions, were used. The experimental design included variation in atmospheric conditions, temperature and time. PLSR was first used to analyse each of the two data types separately in order to explore the effect of the experimental parameters on the data. The results showed that both the gene expression and FT-IR spectra were affected by the variations in atmosphere, temperature and time, but that the effect was different for the two types of data. When the DNA microarray data and FT-IR spectra were linked together by PLSR, covariation due to temperature was seen. Both specific genes and ranges in the FT-IR spectra that were connected to the variation in temperature were detected. Some of these are possibly connected to properties of the cell wall of the bacteria. The results in this study show the potential of PLSR for investigation of covariance structures in biological data. By doing this, valuable information about the biological system can be detected and interpreted. It was also shown that the use of FT-IR spectroscopy provided important information about the stress responses in the bacteria, information that was not detected from the DNA microarray data.

Campylobacter jejuni↗
PubMed2004

A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis.

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.

Animals↗
PubMed2006

Myocyte-specific enhancer factor 2A is essential for zebrafish posterior somite development.

Somite development is governed tightly by genetic factors. In the large-scale mutagenesis screens of zebrafish, no mutations were linked to myocyte enhancer factor 2A (MEF2A) locus. In this study, we find that MEF2A knock-down embryos display a downward tail curvature and have U-shaped posterior somites. Furthermore, we demonstrate that MEF2A is required for Hedgehog signaling. MEF2A inhibition results in induction of apoptosis in the posterior somites. We further find that Hedgehog signaling can negatively regulate MEF2A expression in the somites. Microarray studies reveal a number of genes that are differentially expressed in the MEF2A morphants. Our studies suggest that MEF2A is essential for zebrafish posterior somite development.

Animals↗
PubMed2005

Bioinformatics in otolaryngology.

This article reviews the recent literature on microarray analysis, bioinformatics techniques, and genomics in relation to the study of carcinogenesis of head and neck cancers.

Algorithms↗
PubMed2006

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.

Bacteria↗
PubMed2005

Dark matter in the genome: evidence of widespread transcription detected by microarray tiling experiments.

Microarrays provide the opportunity to measure transcription from regions of the genome without bias towards the location of known genes. This technology thus offers an important source of genomic sequence annotation that is complementary to cDNA sequencing and computational gene-finding methods. Recent "tiling" microarray experiments that assay transcription at regular intervals throughout the genome have shown evidence of large amounts of transcription outside the boundaries of known genes. This transcription is observed in polyadenylated RNA samples and appears to be derived from intergenic regions, from introns of known genes and from sequences antisense to known transcripts. In this article, we discuss different explanations for this phenomenon.

Animals↗
PubMed2005

Molecular analyses of disease pathogenesis: application of bovine microarrays.

The molecular analysis of disease pathogenesis in cattle has been limited by the lack of availability of tools to analyze both host and pathogen responses. These limitations are disappearing with the advent of methodologies such as microarrays that facilitate rapid characterization of global gene expression at the level of individual cells and tissues. The present review focuses on the use of microarray technologies to investigate the functional pathogenomics of infectious disease in cattle. We discuss a number of unique issues that must be addressed when designing both in vitro and in vivo model systems to analyze host responses to a specific pathogen. Furthermore, comparative functional genomic strategies are discussed that can be used to address questions regarding host responses that are either common to a variety of pathogens or unique to individual pathogens. These strategies can also be applied to investigations of cell signaling pathways and the analyses of innate immune responses. Microarray analyses of both host and pathogen responses hold substantial promise for the generation of databases that can be used in the future to address a wide variety of questions. A critical component limiting these comparative analyses will be the quality of the databases and the complete functional annotation of the bovine genome. These limitations are discussed with an indication of future developments that will accelerate the validation of data generated when completing a molecular characterization of disease pathogenesis in cattle.

Animals↗
PubMed2006

A highly standardized, robust, and cost-effective method for genome-wide transcriptome analysis of peripheral blood applicable to large-scale clinical trials.

The use of peripheral blood mononuclear cells (PBMC) for transcriptome analysis has already been proven valuable for assessing disease-associated and drug-response-related gene signatures. While these proof-of-principle studies have been critically important, the instability of RNA within PBMC prohibits their use in large-scale multicenter trials for which samples have to be transported for a prolonged time prior to RNA isolation. Therefore, a prerequisite for transcriptome analysis of peripheral blood in clinical trials will be a standardized and valid method to stabilize the RNA profile immediately after blood withdrawal. Moreover, to be able to perform such large-scale clinical studies routinely in several hundred patients more cost-effective array technologies are required. To address these critical issues, we have combined a whole-blood RNA stabilization technology with a method to reduce globin mRNA, followed by genome-wide transcriptome analysis using a newly introduced BeadChip oligonucleotide technology. We demonstrate that the globin mRNA reduction method results in significantly improved data quality of stabilized RNA samples with low intragroup variance and a detection rate of expressed genes similar to that in PBMC. More important, even small differences in gene expression such as are observed between females and males were detected and sufficient to predict gender in whole-blood samples. We therefore propose the combination of globin mRNA reduction after whole-blood RNA stabilization with a newly introduced cost-effective BeadChip array as the preferred approach for large-scale multicenter trials, especially when establishing predictive markers for disease and treatment outcome in peripheral blood.

Blood Cells↗
PubMed2006

Quality prediction of cell substrate using gene expression profiling.

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.

Adenoviridae↗
PubMed2004

Single-channel quantitative multiplex reverse transcriptase-polymerase chain reaction for large numbers of gene products differentiates nondemented from neuropathological Alzheimer's disease.

Effective approaches using array technologies are critical to understand the molecular bases of human diseases. The results obtained using such procedures require analysis and validation procedures that are still under development. In the context of Alzheimer's disease, in which the identification of molecular mechanisms of underlying pathologies is vital, we describe a robust assay that is the first real-time reverse transcriptase-polymerase chain reaction-based high-throughput approach that can simultaneously quantitate the expression of a large number of genes at the copy number level from a minute amount of starting material. Using this approach within the human brain, we were able to quantitate as many as 19 genes at a time with only one type of fluorescent probe. The number of genes included can be considerably increased. Examples of consistent changes in Alzheimer's disease within these 19 candidate genes included reductions in targets related to the dendritic and synaptic apparatus. These changes were specific to Alzheimer's disease when compared with Parkinson's disease cases. We also present comparison data with microarray analysis from the same brain region and the same patients. The high sensitivity and reproducibility of this technology coupled with appropriate multivariate analysis is proposed here to form a biotechnology platform that can be widely used for diagnostic purposes as well as basic research.

Alzheimer Disease↗
PubMed2003

DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation.

DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12pM. Utilizing a CO(2) IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5pM has been achieved in MHAC devices with sample oscillation.

Carbocyanines↗
PubMed2003

Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.

Cell Division↗