Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

PubMed Central

Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

2014-01-01

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets. PMID:25489607

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

PubMed

Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

2015-02-07

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

Comprehensive performance comparison of high-resolution array platforms for genome-wide Copy Number Variation (CNV) analysis in humans.

PubMed

Haraksingh, Rajini R; Abyzov, Alexej; Urban, Alexander Eckehart

2017-04-24

High-resolution microarray technology is routinely used in basic research and clinical practice to efficiently detect copy number variants (CNVs) across the entire human genome. A new generation of arrays combining high probe densities with optimized designs will comprise essential tools for genome analysis in the coming years. We systematically compared the genome-wide CNV detection power of all 17 available array designs from the Affymetrix, Agilent, and Illumina platforms by hybridizing the well-characterized genome of 1000 Genomes Project subject NA12878 to all arrays, and performing data analysis using both manufacturer-recommended and platform-independent software. We benchmarked the resulting CNV call sets from each array using a gold standard set of CNVs for this genome derived from 1000 Genomes Project whole genome sequencing data. The arrays tested comprise both SNP and aCGH platforms with varying designs and contain between ~0.5 to ~4.6 million probes. Across the arrays CNV detection varied widely in number of CNV calls (4-489), CNV size range (~40 bp to ~8 Mbp), and percentage of non-validated CNVs (0-86%). We discovered strikingly strong effects of specific array design principles on performance. For example, some SNP array designs with the largest numbers of probes and extensive exonic coverage produced a considerable number of CNV calls that could not be validated, compared to designs with probe numbers that are sometimes an order of magnitude smaller. This effect was only partially ameliorated using different analysis software and optimizing data analysis parameters. High-resolution microarrays will continue to be used as reliable, cost- and time-efficient tools for CNV analysis. However, different applications tolerate different limitations in CNV detection. Our study quantified how these arrays differ in total number and size range of detected CNVs as well as sensitivity, and determined how each array balances these attributes. This analysis will inform appropriate array selection for future CNV studies, and allow better assessment of the CNV-analytical power of both published and ongoing array-based genomics studies. Furthermore, our findings emphasize the importance of concurrent use of multiple analysis algorithms and independent experimental validation in array-based CNV detection studies.

Normal uniform mixture differential gene expression detection for cDNA microarrays

PubMed Central

Dean, Nema; Raftery, Adrian E

2005-01-01

Background One of the primary tasks in analysing gene expression data is finding genes that are differentially expressed in different samples. Multiple testing issues due to the thousands of tests run make some of the more popular methods for doing this problematic. Results We propose a simple method, Normal Uniform Differential Gene Expression (NUDGE) detection for finding differentially expressed genes in cDNA microarrays. The method uses a simple univariate normal-uniform mixture model, in combination with new normalization methods for spread as well as mean that extend the lowess normalization of Dudoit, Yang, Callow and Speed (2002) [1]. It takes account of multiple testing, and gives probabilities of differential expression as part of its output. It can be applied to either single-slide or replicated experiments, and it is very fast. Three datasets are analyzed using NUDGE, and the results are compared to those given by other popular methods: unadjusted and Bonferroni-adjusted t tests, Significance Analysis of Microarrays (SAM), and Empirical Bayes for microarrays (EBarrays) with both Gamma-Gamma and Lognormal-Normal models. Conclusion The method gives a high probability of differential expression to genes known/suspected a priori to be differentially expressed and a low probability to the others. In terms of known false positives and false negatives, the method outperforms all multiple-replicate methods except for the Gamma-Gamma EBarrays method to which it offers comparable results with the added advantages of greater simplicity, speed, fewer assumptions and applicability to the single replicate case. An R package called nudge to implement the methods in this paper will be made available soon at . PMID:16011807

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

PubMed

Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

2007-01-24

An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

DNA Microarray Detection of 18 Important Human Blood Protozoan Species

PubMed Central

Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

2016-01-01

Background Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. Methodology/Principal Findings The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Conclusions/Significance Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species. PMID:27911895

SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration.

PubMed

Parro, Víctor; de Diego-Castilla, Graciela; Rodríguez-Manfredi, José A; Rivas, Luis A; Blanco-López, Yolanda; Sebastián, Eduardo; Romeral, Julio; Compostizo, Carlos; Herrero, Pedro L; García-Marín, Adolfo; Moreno-Paz, Mercedes; García-Villadangos, Miriam; Cruz-Gil, Patricia; Peinado, Verónica; Martín-Soler, Javier; Pérez-Mercader, Juan; Gómez-Elvira, Javier

2011-01-01

The search for unequivocal signs of life on other planetary bodies is one of the major challenges for astrobiology. The failure to detect organic molecules on the surface of Mars by measuring volatile compounds after sample heating, together with the new knowledge of martian soil chemistry, has prompted the astrobiological community to develop new methods and technologies. Based on protein microarray technology, we have designed and built a series of instruments called SOLID (for "Signs Of LIfe Detector") for automatic in situ detection and identification of substances or analytes from liquid and solid samples (soil, sediments, or powder). Here, we present the SOLID3 instrument, which is able to perform both sandwich and competitive immunoassays and consists of two separate functional units: a Sample Preparation Unit (SPU) for 10 different extractions by ultrasonication and a Sample Analysis Unit (SAU) for fluorescent immunoassays. The SAU consists of five different flow cells, with an antibody microarray in each one (2000 spots). It is also equipped with an exclusive optical package and a charge-coupled device (CCD) for fluorescent detection. We demonstrated the performance of SOLID3 in the detection of a broad range of molecular-sized compounds, which range from peptides and proteins to whole cells and spores, with sensitivities at 1-2 ppb (ng mL⁻¹) for biomolecules and 10⁴ to 10³ spores per milliliter. We report its application in the detection of acidophilic microorganisms in the Río Tinto Mars analogue and report the absence of substantial negative effects on the immunoassay in the presence of 50 mM perchlorate (20 times higher than that found at the Phoenix landing site). Our SOLID instrument concept is an excellent option with which to detect biomolecules because it avoids the high-temperature treatments that may destroy organic matter in the presence of martian oxidants.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

PubMed

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong; Li, Ning

2016-12-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma

PubMed Central

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong

2016-01-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC (n = 65) and healthy control subjects (n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC. PMID:27885040

Polysaccharide Microarray Technology for the Detection of Burkholderia Pseudomallei and Burkholderia Mallei Antibodies

DTIC Science & Technology

2006-04-27

polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides . This... polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray... Polysaccharide microarrays; Burkholderia pseudomallei; Burkholderia mallei; Glanders; Melioidosis1. Introduction There has been a great deal of emphasis on the

Development and Comparison of Two Assay Formats for Parallel Detection of Four Biothreat Pathogens by Using Suspension Microarrays

PubMed Central

Janse, Ingmar; Bok, Jasper M.; Hamidjaja, Raditijo A.; Hodemaekers, Hennie M.; van Rotterdam, Bart J.

2012-01-01

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics. PMID:22355407

Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays.

PubMed

Janse, Ingmar; Bok, Jasper M; Hamidjaja, Raditijo A; Hodemaekers, Hennie M; van Rotterdam, Bart J

2012-01-01

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.

An Introduction to MAMA (Meta-Analysis of MicroArray data) System.

PubMed

Zhang, Zhe; Fenstermacher, David

2005-01-01

Analyzing microarray data across multiple experiments has been proven advantageous. To support this kind of analysis, we are developing a software system called MAMA (Meta-Analysis of MicroArray data). MAMA utilizes a client-server architecture with a relational database on the server-side for the storage of microarray datasets collected from various resources. The client-side is an application running on the end user's computer that allows the user to manipulate microarray data and analytical results locally. MAMA implementation will integrate several analytical methods, including meta-analysis within an open-source framework offering other developers the flexibility to plug in additional statistical algorithms.

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

PubMed Central

Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

2012-01-01

We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species.

PubMed

Ahn, Soohyoun; Kulis, David M; Erdner, Deana L; Anderson, Donald M; Walt, David R

2006-09-01

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 mum) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.

MMASS: an optimized array-based method for assessing CpG island methylation.

PubMed

Ibrahim, Ashraf E K; Thorne, Natalie P; Baird, Katie; Barbosa-Morais, Nuno L; Tavaré, Simon; Collins, V Peter; Wyllie, Andrew H; Arends, Mark J; Brenton, James D

2006-01-01

We describe an optimized microarray method for identifying genome-wide CpG island methylation called microarray-based methylation assessment of single samples (MMASS) which directly compares methylated to unmethylated sequences within a single sample. To improve previous methods we used bioinformatic analysis to predict an optimized combination of methylation-sensitive enzymes that had the highest utility for CpG-island probes and different methods to produce unmethylated representations of test DNA for more sensitive detection of differential methylation by hybridization. Subtraction or methylation-dependent digestion with McrBC was used with optimized (MMASS-v2) or previously described (MMASS-v1, MMASS-sub) methylation-sensitive enzyme combinations and compared with a published McrBC method. Comparison was performed using DNA from the cell line HCT116. We show that the distribution of methylation microarray data is inherently skewed and requires exogenous spiked controls for normalization and that analysis of digestion of methylated and unmethylated control sequences together with linear fit models of replicate data showed superior statistical power for the MMASS-v2 method. Comparison with previous methylation data for HCT116 and validation of CpG islands from PXMP4, SFRP2, DCC, RARB and TSEN2 confirmed the accuracy of MMASS-v2 results. The MMASS-v2 method offers improved sensitivity and statistical power for high-throughput microarray identification of differential methylation.

Development of a low-cost detection method for miRNA microarray.

PubMed

Li, Wei; Zhao, Botao; Jin, Youxin; Ruan, Kangcheng

2010-04-01

MicroRNA (miRNA) microarray is a powerful tool to explore the expression profiling of miRNA. The current detection method used in miRNA microarray is mainly fluorescence based, which usually requires costly detection system such as laser confocal scanner of tens of thousands of dollars. Recently, we developed a low-cost yet sensitive detection method for miRNA microarray based on enzyme-linked assay. In this approach, the biotinylated miRNAs were captured by the corresponding oligonucleotide probes immobilized on microarray slide; and then the biotinylated miRNAs would capture streptavidin-conjugated alkaline phosphatase. A purple-black precipitation on each biotinylated miRNA spot was produced by the enzyme catalytic reaction. It could be easily detected by a charge-coupled device digital camera mounted on a microscope, which lowers the detection cost more than 100 fold compared with that of fluorescence method. Our data showed that signal intensity of the spot correlates well with the biotinylated miRNA concentration and the detection limit for miRNAs is at least 0.4 fmol and the detection dynamic range spans about 2.5 orders of magnitude, which is comparable to that of fluorescence method.

Evaluation of microarray data normalization procedures using spike-in experiments

PubMed Central

Rydén, Patrik; Andersson, Henrik; Landfors, Mattias; Näslund, Linda; Hartmanová, Blanka; Noppa, Laila; Sjöstedt, Anders

2006-01-01

Background Recently, a large number of methods for the analysis of microarray data have been proposed but there are few comparisons of their relative performances. By using so-called spike-in experiments, it is possible to characterize the analyzed data and thereby enable comparisons of different analysis methods. Results A spike-in experiment using eight in-house produced arrays was used to evaluate established and novel methods for filtration, background adjustment, scanning, channel adjustment, and censoring. The S-plus package EDMA, a stand-alone tool providing characterization of analyzed cDNA-microarray data obtained from spike-in experiments, was developed and used to evaluate 252 normalization methods. For all analyses, the sensitivities at low false positive rates were observed together with estimates of the overall bias and the standard deviation. In general, there was a trade-off between the ability of the analyses to identify differentially expressed genes (i.e. the analyses' sensitivities) and their ability to provide unbiased estimators of the desired ratios. Virtually all analysis underestimated the magnitude of the regulations; often less than 50% of the true regulations were observed. Moreover, the bias depended on the underlying mRNA-concentration; low concentration resulted in high bias. Many of the analyses had relatively low sensitivities, but analyses that used either the constrained model (i.e. a procedure that combines data from several scans) or partial filtration (a novel method for treating data from so-called not-found spots) had with few exceptions high sensitivities. These methods gave considerable higher sensitivities than some commonly used analysis methods. Conclusion The use of spike-in experiments is a powerful approach for evaluating microarray preprocessing procedures. Analyzed data are characterized by properties of the observed log-ratios and the analysis' ability to detect differentially expressed genes. If bias is not a major problem; we recommend the use of either the CM-procedure or partial filtration. PMID:16774679

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies.

PubMed

Parthasarathy, Narayanan; DeShazer, David; England, Marilyn; Waag, David M

2006-11-01

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.

EDRN Biomarker Reference Lab: Pacific Northwest National Laboratory — EDRN Public Portal

Cancer.gov

The purpose of this project is to develop antibody microarrays incorporating three major improvements compared to previous antibody microarray platforms, and to produce and disseminate these antibody microarray technologies for the Early Detection Research Network (EDRN) and the research community focusing on early detection, and risk assessment of cancer.

The SOLID (Signs Of LIfe Detector) instrument concept: an antibody microarray-based biosensor for life detection in astrobiology

NASA Astrophysics Data System (ADS)

Parro, V.; Rivas, L. A.; Rodríguez-Manfredi, J. A.; Blanco, Y.; de Diego-Castilla, G.; Cruz-Gil, P.; Moreno-Paz, M.; García-Villadangos, M.; Compostizo, C.; Herrero, P. L.

2009-04-01

Immunosensors have been extensively used since many years for environmental monitoring. Different technological platforms allow new biosensor designs and implementations. We have reported (Rivas et al., 2008) a shotgun approach for antibody production for biomarker detection in astrobiology and environmental monitoring, the production of 150 new polyclonal antibodies against microbial strains and environmental extracts, and the construction and validation of an antibody microarray (LDCHIP200, for "Life Detector Chip") containing 200 different antibodies. We have successfully used the LDCHIP200 for the detection of biological polymers in extreme environments in different parts of the world (e.g., a deep South African mine, Antarctica's Dry valleys, Yellowstone, Iceland, and Rio Tinto). Clustering analysis associated similar immunopatterns to samples from apparently very different environments, indicating that they indeed share similar universal biomarkers. A redundancy in the number of antibodies against different target biomarkers apart of revealing the presence of certain biomolecules, it renders a sample-specific immuno-profile, an "immnuno-fingerprint", which may constitute by itself an indirect biosignature. We will present a case study of immunoprofiling different iron-sulfur as well as phylosilicates rich samples along the Rio Tinto river banks. Based on protein microarray technology, we designed and built the concept instrument called SOLID (for "Signs Of LIfe Detector"; Parro et al., 2005; 2008a, b; http://cab.inta.es/solid) for automatic in situ analysis of soil samples and molecular biomarkers detection. A field prototype, SOLID2, was successfully tested for the analysis of grinded core samples during the 2005 "MARTE" campaign of a Mars drilling simulation experiment by a sandwich microarray immunoassay (Parro et al., 2008b). We will show the new version of the instrument (SOLID3) which is able to perform both sandwich and competitive immunoassays. SOLID3 consists of two separate functional units: a Sample Preparation Unit (SPU), for ten different extractions by ultrasonication, and a Sample Analysis Unit (SAU), for fluorescent immunoassays. The SAU consists of ten different flow cells each of one allocate one antibody microarray (up to 2000 spots), and is equipped with an unique designed optical package for fluorescent detection. We demonstrate the performance of SOLID3 for the detection of a broad range of molecular size compounds, from the amino acid size, peptides, proteins, to whole cells and spores, with sensitivities at the ppb level. References Parro, V., et al., 2005. Planetary and Space Science 53: 729-737. Parro, V., et al., 2008a. Space Science Reviews 135: 293-311 Parro, V., et al., 2008b. Astrobiology 8:987-99 Rivas, L. A., et al., 2008. Analytical Chemistry 80: 7970-7979

Unraveling transcriptional control and cis-regulatory codes using the software suite GeneACT

PubMed Central

Cheung, Tom Hiu; Kwan, Yin Lam; Hamady, Micah; Liu, Xuedong

2006-01-01

Deciphering gene regulatory networks requires the systematic identification of functional cis-acting regulatory elements. We present a suite of web-based bioinformatics tools, called GeneACT , that can rapidly detect evolutionarily conserved transcription factor binding sites or microRNA target sites that are either unique or over-represented in differentially expressed genes from DNA microarray data. GeneACT provides graphic visualization and extraction of common regulatory sequence elements in the promoters and 3'-untranslated regions that are conserved across multiple mammalian species. PMID:17064417

A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnum, Susan M.

An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

Reusable conductimetric array of interdigitated microelectrodes for the readout of low-density microarrays.

PubMed

Mallén, Maria; Díaz-González, María; Bonilla, Diana; Salvador, Juan P; Marco, María P; Baldi, Antoni; Fernández-Sánchez, César

2014-06-17

Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0 ng mL(-1), this being below the threshold value set by the World Anti-Doping Agency and the European Community. Copyright © 2014 Elsevier B.V. All rights reserved.

The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

PubMed

Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

2017-05-01

Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

PubMed

Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R

2009-07-22

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

PubMed Central

Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

2013-01-01

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

PubMed

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

PubMed

Parthasarathy, N; Saksena, R; Kováč, P; Deshazer, D; Peacock, S J; Wuthiekanun, V; Heine, H S; Friedlander, A M; Cote, C K; Welkos, S L; Adamovicz, J J; Bavari, S; Waag, D M

2008-11-03

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

PubMed

Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

2016-07-15

Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Development and application of antibody microarray for lymphocystis disease virus detection in fish.

PubMed

Sheng, Xiuzhen; Xu, Xiaoli; Zhan, Wenbin

2013-05-01

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3-conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-L-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55μg/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 μg/ml when streptavidin-biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at -20°C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n=50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish. Copyright © 2013 Elsevier B.V. All rights reserved.

A new measure for gene expression biclustering based on non-parametric correlation.

PubMed

Flores, Jose L; Inza, Iñaki; Larrañaga, Pedro; Calvo, Borja

2013-12-01

One of the emerging techniques for performing the analysis of the DNA microarray data known as biclustering is the search of subsets of genes and conditions which are coherently expressed. These subgroups provide clues about the main biological processes. Until now, different approaches to this problem have been proposed. Most of them use the mean squared residue as quality measure but relevant and interesting patterns can not be detected such as shifting, or scaling patterns. Furthermore, recent papers show that there exist new coherence patterns involved in different kinds of cancer and tumors such as inverse relationships between genes which can not be captured. The proposed measure is called Spearman's biclustering measure (SBM) which performs an estimation of the quality of a bicluster based on the non-linear correlation among genes and conditions simultaneously. The search of biclusters is performed by using a evolutionary technique called estimation of distribution algorithms which uses the SBM measure as fitness function. This approach has been examined from different points of view by using artificial and real microarrays. The assessment process has involved the use of quality indexes, a set of bicluster patterns of reference including new patterns and a set of statistical tests. It has been also examined the performance using real microarrays and comparing to different algorithmic approaches such as Bimax, CC, OPSM, Plaid and xMotifs. SBM shows several advantages such as the ability to recognize more complex coherence patterns such as shifting, scaling and inversion and the capability to selectively marginalize genes and conditions depending on the statistical significance. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Microarray analysis of the rat lacrimal gland following the loss of parasympathetic control of secretion

PubMed Central

Nguyen, Doan H.; Toshida, Hiroshi; Schurr, Jill; Beuerman, Roger W.

2010-01-01

Previous studies showed that loss of muscarinic parasympathetic input to the lacrimal gland (LG) leads to a dramatic reduction in tear secretion and profound changes to LG structure. In this study, we used DNA microarrays to examine the regulation of the gene expression of the genes for secretory function and organization of the LG. Long-Evans rats anesthetized with a mixture of ketamine/xylazine (80:10 mg/kg) underwent unilateral sectioning of the greater superficial petrosal nerve, the input to the pterygopalatine ganglion. After 7 days, tear secretion was measured, the animals were killed, and structural changes in the LG were examined by light microscopy. Total RNA from control and experimental LGs (n = 5) was used for DNA microarray analysis employing the U34A GeneChip. Three statistical algorithms (detection, change call, and signal log ratio) were used to determine differential gene expression using the Microarray Suite (5.0) and Data Mining Tools (3.0). Tear secretion was significantly reduced and corneal ulcers developed in all experimental eyes. Light microscopy showed breakdown of the acinar structure of the LG. DNA microarray analysis showed downregulation of genes associated with the endoplasmic reticulum and Golgi, including genes involved in protein folding and processing. Conversely, transcripts for cytoskeleton and extracellular matrix components, inflammation, and apoptosis were upregulated. The number of significantly upregulated genes (116) was substantially greater than the number of downregulated genes (49). Removal of the main secretory input to the rat LG resulted in clinical symptoms associated with severe dry eye. Components of the secretory pathway were negatively affected, and the increase in cell proliferation and inflammation may lead to loss of organization in the parasympathectomized lacrimal gland. PMID:15084711

4p terminal deletion and 11p subtelomeric duplication detected by genomic microarray in a patient with Wolf-Hirschhorn syndrome and an atypical phenotype.

PubMed

Stevenson, David A; Carey, John C; Cowley, Brett C; Bayrak-Toydemir, Pinar; Mao, Rong; Brothman, Arthur R

2004-12-01

We report a de novo cryptic 11p duplication found by genomic microarray with a cytogenetically detected 4p deletion. Terminal 4p deletions cause Wolf-Hirschhorn syndrome, but the phenotype probably was modified by the paternally derived 11p duplication. This emphasizes the clinical utility of genomic microarray.

mRNA-Based Parallel Detection of Active Methanotroph Populations by Use of a Diagnostic Microarray

PubMed Central

Bodrossy, Levente; Stralis-Pavese, Nancy; Konrad-Köszler, Marianne; Weilharter, Alexandra; Reichenauer, Thomas G.; Schöfer, David; Sessitsch, Angela

2006-01-01

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities. PMID:16461725

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

PubMed

Erickson, A; Fisher, M; Furukawa-Stoffer, T; Ambagala, A; Hodko, D; Pasick, J; King, D P; Nfon, C; Ortega Polo, R; Lung, O

2018-04-01

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases. © 2017 Her Majesty the Queen in Right of Canada • Transboundary and Emerging Diseases.

DNA Microarray for Rapid Detection and Identification of Food and Water Borne Bacteria: From Dry to Wet Lab.

PubMed

Ranjbar, Reza; Behzadi, Payam; Najafi, Ali; Roudi, Raheleh

2017-01-01

A rapid, accurate, flexible and reliable diagnostic method may significantly decrease the costs of diagnosis and treatment. Designing an appropriate microarray chip reduces noises and probable biases in the final result. The aim of this study was to design and construct a DNA Microarray Chip for a rapid detection and identification of 10 important bacterial agents. In the present survey, 10 unique genomic regions relating to 10 pathogenic bacterial agents including Escherichia coli (E.coli), Shigella boydii, Sh.dysenteriae, Sh.flexneri, Sh.sonnei, Salmonella typhi, S.typhimurium, Brucella sp., Legionella pneumophila, and Vibrio cholera were selected for designing specific long oligo microarray probes. For this reason, the in-silico operations including utilization of the NCBI RefSeq database, Servers of PanSeq and Gview, AlleleID 7.7 and Oligo Analyzer 3.1 was done. On the other hand, the in-vitro part of the study comprised stages of robotic microarray chip probe spotting, bacterial DNAs extraction and DNA labeling, hybridization and microarray chip scanning. In wet lab section, different tools and apparatus such as Nexterion® Slide E, Qarray mini spotter, NimbleGen kit, TrayMix TM S4, and Innoscan 710 were used. A DNA microarray chip including 10 long oligo microarray probes was designed and constructed for detection and identification of 10 pathogenic bacteria. The DNA microarray chip was capable to identify all 10 bacterial agents tested simultaneously. The presence of a professional bioinformatician as a probe designer is needed to design appropriate multifunctional microarray probes to increase the accuracy of the outcomes.

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels.

PubMed

Sevenler, Derin; Daaboul, George G; Ekiz Kanik, Fulya; Ünlü, Neşe Lortlar; Ünlü, M Selim

2018-05-21

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

PubMed Central

Li, Xiang; Harwood, Valerie J.; Nayak, Bina

2016-01-01

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. PMID:26729716

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

PubMed

Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

2004-12-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

Development of a single nucleotide polymorphism DNA microarray for the detection and genotyping of the SARS coronavirus.

PubMed

Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin

2014-10-01

Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.

Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

EPA Science Inventory

Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

Employing image processing techniques for cancer detection using microarray images.

PubMed

Dehghan Khalilabad, Nastaran; Hassanpour, Hamid

2017-02-01

Microarray technology is a powerful genomic tool for simultaneously studying and analyzing the behavior of thousands of genes. The analysis of images obtained from this technology plays a critical role in the detection and treatment of diseases. The aim of the current study is to develop an automated system for analyzing data from microarray images in order to detect cancerous cases. The proposed system consists of three main phases, namely image processing, data mining, and the detection of the disease. The image processing phase performs operations such as refining image rotation, gridding (locating genes) and extracting raw data from images the data mining includes normalizing the extracted data and selecting the more effective genes. Finally, via the extracted data, cancerous cell is recognized. To evaluate the performance of the proposed system, microarray database is employed which includes Breast cancer, Myeloid Leukemia and Lymphomas from the Stanford Microarray Database. The results indicate that the proposed system is able to identify the type of cancer from the data set with an accuracy of 95.45%, 94.11%, and 100%, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improved porous silicon (P-Si) microarray based PSA (prostate specific antigen) immunoassay by optimized surface density of the capture antibody

PubMed Central

Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

2014-01-01

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL−1, 80pgmL−1, and 800fgmL−1 when arraying the PSA antibody, H117 at the concentration 15µgmL−1, 35µgmL−1 and 154µgmL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL−1 to 500ngmL−1. The microarray showed a LOD of 800fgmL−1 and a dynamic range of 800 fgmL−1 to 80ngmL−1 in serum spiked samples. PMID:24016590

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units.

PubMed

Blom, H; Gösch, M

2004-04-01

The past few years we have witnessed a tremendous surge of interest in so-called array-based miniaturised analytical systems due to their value as extremely powerful tools for high-throughput sequence analysis, drug discovery and development, and diagnostic tests in medicine (see articles in Issue 1). Terminologies that have been used to describe these array-based bioscience systems include (but are not limited to): DNA-chip, microarrays, microchip, biochip, DNA-microarrays and genome chip. Potential technological benefits of introducing these miniaturised analytical systems include improved accuracy, multiplexing, lower sample and reagent consumption, disposability, and decreased analysis times, just to mention a few examples. Among the many alternative principles of detection-analysis (e.g.chemiluminescence, electroluminescence and conductivity), fluorescence-based techniques are widely used, examples being fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarisation, time-resolved fluorescence, and fluorescence fluctuation spectroscopy (see articles in Issue 11). Time-dependent fluctuations of fluorescent biomolecules with different molecular properties, like molecular weight, translational and rotational diffusion time, colour and lifetime, potentially provide all the kinetic and thermodynamic information required in analysing complex interactions. In this mini-review article, we present recent extensions aimed to implement parallel laser excitation and parallel fluorescence detection that can lead to even further increase in throughput in miniaturised array-based analytical systems. We also report on developments and characterisations of multiplexing extension that allow multifocal laser excitation together with matched parallel fluorescence detection for parallel confocal dynamical fluorescence fluctuation studies at the single biomolecule level.

Parallel evaluation of broad virus detection methods.

PubMed

Modrof, Jens; Berting, Andreas; Kreil, Thomas R

2014-01-01

The testing for adventitious viruses is of critical importance during development and production of biological products. The recent emergence and ongoing development of broad virus detection methods calls for an evaluation of whether these methods can appropriately be implemented into current adventitious agent testing procedures. To assess the suitability of several broad virus detection methods, a comparative experimental study was conducted: four virus preparations, which were spiked at two different concentrations each into two different cell culture media, were sent to four investigators in a blinded fashion for analysis with broad virus detection methods such as polymerase chain reaction-electrospray ionization mass spectrometry (PCR-ESI/MS), microarray, and two approaches utilizing massively parallel sequencing. The results that were reported by the investigators revealed that all methods were able to identify the majority of samples correctly (mean 83%), with a surprisingly narrow range among the methods, that is, between 72% (PCR-ESI/MS) and 95% (microarray). In addition to the correct results, a variety of unexpected assignments were reported for a minority of samples, again with little variation regarding the methods used (range 20-45%), while false negatives were reported for 0-25% of the samples. Regarding assay sensitivity, the viruses were detected by all methods included in this study at concentrations of about 4-5 log10 quantitative PCR copies/mL, and probably with higher sensitivity in some cases. In summary, the broad virus detection methods investigated were shown to be suitable even for detection of relatively low virus concentrations. However, there is also some potential for the production of false-positive as well as false-negative assignments, which indicates the requirement for further improvements before these methods can be considered for routine use. © PDA, Inc. 2014.

Reverse phase protein microarrays: fluorometric and colorimetric detection.

PubMed

Gallagher, Rosa I; Silvestri, Alessandra; Petricoin, Emanuel F; Liotta, Lance A; Espina, Virginia

2011-01-01

The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

Microfluidic microarray systems and methods thereof

DOEpatents

West, Jay A. A. [Castro Valley, CA; Hukari, Kyle W [San Ramon, CA; Hux, Gary A [Tracy, CA

2009-04-28

Disclosed are systems that include a manifold in fluid communication with a microfluidic chip having a microarray, an illuminator, and a detector in optical communication with the microarray. Methods for using these systems for biological detection are also disclosed.

A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

PubMed

Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

2014-04-25

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

PubMed Central

Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

2014-01-01

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

An integrated approach for identifying wrongly labelled samples when performing classification in microarray data.

PubMed

Leung, Yuk Yee; Chang, Chun Qi; Hung, Yeung Sam

2012-01-01

Using hybrid approach for gene selection and classification is common as results obtained are generally better than performing the two tasks independently. Yet, for some microarray datasets, both classification accuracy and stability of gene sets obtained still have rooms for improvement. This may be due to the presence of samples with wrong class labels (i.e. outliers). Outlier detection algorithms proposed so far are either not suitable for microarray data, or only solve the outlier detection problem on their own. We tackle the outlier detection problem based on a previously proposed Multiple-Filter-Multiple-Wrapper (MFMW) model, which was demonstrated to yield promising results when compared to other hybrid approaches (Leung and Hung, 2010). To incorporate outlier detection and overcome limitations of the existing MFMW model, three new features are introduced in our proposed MFMW-outlier approach: 1) an unbiased external Leave-One-Out Cross-Validation framework is developed to replace internal cross-validation in the previous MFMW model; 2) wrongly labeled samples are identified within the MFMW-outlier model; and 3) a stable set of genes is selected using an L1-norm SVM that removes any redundant genes present. Six binary-class microarray datasets were tested. Comparing with outlier detection studies on the same datasets, MFMW-outlier could detect all the outliers found in the original paper (for which the data was provided for analysis), and the genes selected after outlier removal were proven to have biological relevance. We also compared MFMW-outlier with PRAPIV (Zhang et al., 2006) based on same synthetic datasets. MFMW-outlier gave better average precision and recall values on three different settings. Lastly, artificially flipped microarray datasets were created by removing our detected outliers and flipping some of the remaining samples' labels. Almost all the 'wrong' (artificially flipped) samples were detected, suggesting that MFMW-outlier was sufficiently powerful to detect outliers in high-dimensional microarray datasets.

Identification and handling of artifactual gene expression profiles emerging in microarray hybridization experiments

PubMed Central

Brodsky, Leonid; Leontovich, Andrei; Shtutman, Michael; Feinstein, Elena

2004-01-01

Mathematical methods of analysis of microarray hybridizations deal with gene expression profiles as elementary units. However, some of these profiles do not reflect a biologically relevant transcriptional response, but rather stem from technical artifacts. Here, we describe two technically independent but rationally interconnected methods for identification of such artifactual profiles. Our diagnostics are based on detection of deviations from uniformity, which is assumed as the main underlying principle of microarray design. Method 1 is based on detection of non-uniformity of microarray distribution of printed genes that are clustered based on the similarity of their expression profiles. Method 2 is based on evaluation of the presence of gene-specific microarray spots within the slides’ areas characterized by an abnormal concentration of low/high differential expression values, which we define as ‘patterns of differentials’. Applying two novel algorithms, for nested clustering (method 1) and for pattern detection (method 2), we can make a dual estimation of the profile’s quality for almost every printed gene. Genes with artifactual profiles detected by method 1 may then be removed from further analysis. Suspicious differential expression values detected by method 2 may be either removed or weighted according to the probabilities of patterns that cover them, thus diminishing their input in any further data analysis. PMID:14999086

A microarray immunoassay for simultaneous detection of proteins and bacteria

NASA Technical Reports Server (NTRS)

Delehanty, James B.; Ligler, Frances S.

2002-01-01

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus

PubMed Central

Price, Jordan V.; Haddon, David J.; Kemmer, Dodge; Delepine, Guillaume; Mandelbaum, Gil; Jarrell, Justin A.; Gupta, Rohit; Balboni, Imelda; Chakravarty, Eliza F.; Sokolove, Jeremy; Shum, Anthony K.; Anderson, Mark S.; Cheng, Mickie H.; Robinson, William H.; Browne, Sarah K.; Holland, Steven M.; Baechler, Emily C.; Utz, Paul J.

2013-01-01

Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor–binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor–binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell–activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α–driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE. PMID:24270423

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

PubMed Central

Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

2011-01-01

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

Split-plot microarray experiments: issues of design, power and sample size.

PubMed

Tsai, Pi-Wen; Lee, Mei-Ling Ting

2005-01-01

This article focuses on microarray experiments with two or more factors in which treatment combinations of the factors corresponding to the samples paired together onto arrays are not completely random. A main effect of one (or more) factor(s) is confounded with arrays (the experimental blocks). This is called a split-plot microarray experiment. We utilise an analysis of variance (ANOVA) model to assess differentially expressed genes for between-array and within-array comparisons that are generic under a split-plot microarray experiment. Instead of standard t- or F-test statistics that rely on mean square errors of the ANOVA model, we use a robust method, referred to as 'a pooled percentile estimator', to identify genes that are differentially expressed across different treatment conditions. We illustrate the design and analysis of split-plot microarray experiments based on a case application described by Jin et al. A brief discussion of power and sample size for split-plot microarray experiments is also presented.

Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

PubMed

Szkola, A; Linares, E M; Worbs, S; Dorner, B G; Dietrich, R; Märtlbauer, E; Niessner, R; Seidel, M

2014-11-21

Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 μg L(-1) for ricin, 0.1 ± 0.1 μg L(-1) for SEB and 2.3 ± 1.7 μg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

PubMed

Strauss, Christian; Endimiani, Andrea; Perreten, Vincent

2015-01-01

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance. Copyright © 2014 Elsevier B.V. All rights reserved.

Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

PubMed Central

Grubaugh, Nathan D.; McMenamy, Scott S.; Turell, Michael J.; Lee, John S.

2013-01-01

Background Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae). Methodology/Principal Findings The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. Conclusions/Significance We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs. PMID:23967358

Genotyping microarray: Mutation screening in Spanish families with autosomal dominant retinitis pigmentosa

PubMed Central

García-Hoyos, María; Cortón, Marta; Ávila-Fernández, Almudena; Riveiro-Álvarez, Rosa; Giménez, Ascensión; Hernan, Inma; Carballo, Miguel; Ayuso, Carmen

2012-01-01

Purpose Presently, 22 genes have been described in association with autosomal dominant retinitis pigmentosa (adRP); however, they explain only 50% of all cases, making genetic diagnosis of this disease difficult and costly. The aim of this study was to evaluate a specific genotyping microarray for its application to the molecular diagnosis of adRP in Spanish patients. Methods We analyzed 139 unrelated Spanish families with adRP. Samples were studied by using a genotyping microarray (adRP). All mutations found were further confirmed with automatic sequencing. Rhodopsin (RHO) sequencing was performed in all negative samples for the genotyping microarray. Results The adRP genotyping microarray detected the mutation associated with the disease in 20 of the 139 families with adRP. As in other populations, RHO was found to be the most frequently mutated gene in these families (7.9% of the microarray genotyped families). The rate of false positives (microarray results not confirmed with sequencing) and false negatives (mutations in RHO detected with sequencing but not with the genotyping microarray) were established, and high levels of analytical sensitivity (95%) and specificity (100%) were found. Diagnostic accuracy was 15.1%. Conclusions The adRP genotyping microarray is a quick, cost-efficient first step in the molecular diagnosis of Spanish patients with adRP. PMID:22736939

Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

PubMed

Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

2016-05-15

Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously. Copyright © 2015 Elsevier B.V. All rights reserved.

See what you eat--broad GMO screening with microarrays.

PubMed

von Götz, Franz

2010-03-01

Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative.

Improving cluster-based missing value estimation of DNA microarray data.

PubMed

Brás, Lígia P; Menezes, José C

2007-06-01

We present a modification of the weighted K-nearest neighbours imputation method (KNNimpute) for missing values (MVs) estimation in microarray data based on the reuse of estimated data. The method was called iterative KNN imputation (IKNNimpute) as the estimation is performed iteratively using the recently estimated values. The estimation efficiency of IKNNimpute was assessed under different conditions (data type, fraction and structure of missing data) by the normalized root mean squared error (NRMSE) and the correlation coefficients between estimated and true values, and compared with that of other cluster-based estimation methods (KNNimpute and sequential KNN). We further investigated the influence of imputation on the detection of differentially expressed genes using SAM by examining the differentially expressed genes that are lost after MV estimation. The performance measures give consistent results, indicating that the iterative procedure of IKNNimpute can enhance the prediction ability of cluster-based methods in the presence of high missing rates, in non-time series experiments and in data sets comprising both time series and non-time series data, because the information of the genes having MVs is used more efficiently and the iterative procedure allows refining the MV estimates. More importantly, IKNN has a smaller detrimental effect on the detection of differentially expressed genes.

Call for a Computer-Aided Cancer Detection and Classification Research Initiative in Oman.

PubMed

Mirzal, Andri; Chaudhry, Shafique Ahmad

2016-01-01

Cancer is a major health problem in Oman. It is reported that cancer incidence in Oman is the second highest after Saudi Arabia among Gulf Cooperation Council countries. Based on GLOBOCAN estimates, Oman is predicted to face an almost two-fold increase in cancer incidence in the period 2008-2020. However, cancer research in Oman is still in its infancy. This is due to the fact that medical institutions and infrastructure that play central roles in data collection and analysis are relatively new developments in Oman. We believe the country requires an organized plan and efforts to promote local cancer research. In this paper, we discuss current research progress in cancer diagnosis using machine learning techniques to optimize computer aided cancer detection and classification (CAD). We specifically discuss CAD using two major medical data, i.e., medical imaging and microarray gene expression profiling, because medical imaging like mammography, MRI, and PET have been widely used in Oman for assisting radiologists in early cancer diagnosis and microarray data have been proven to be a reliable source for differential diagnosis. We also discuss future cancer research directions and benefits to Oman economy for entering the cancer research and treatment business as it is a multi-billion dollar industry worldwide.

Dynamic variable selection in SNP genotype autocalling from APEX microarray data.

PubMed

Podder, Mohua; Welch, William J; Zamar, Ruben H; Tebbutt, Scott J

2006-11-30

Single nucleotide polymorphisms (SNPs) are DNA sequence variations, occurring when a single nucleotide--adenine (A), thymine (T), cytosine (C) or guanine (G)--is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX). This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart) is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias. Using a set of 32 Coriell DNA samples plus three negative PCR controls as a training data set, we have developed a fully-automated genotyping algorithm based on simple linear discriminant analysis (LDA) using dynamic variable selection. The algorithm combines separate analyses based on the multiple probe sets to give a final posterior probability for each candidate genotype. We have tested our algorithm on a completely independent data set of 270 DNA samples, with validated genotypes, from patients admitted to the intensive care unit (ICU) of St. Paul's Hospital (plus one negative PCR control sample). Our method achieves a concordance rate of 98.9% with a 99.6% call rate for a set of 96 SNPs. By adjusting the threshold value for the final posterior probability of the called genotype, the call rate reduces to 94.9% with a higher concordance rate of 99.6%. We also reversed the two independent data sets in their training and testing roles, achieving a concordance rate up to 99.8%. The strength of this APEX chemistry-based platform is its unique redundancy having multiple probes for a single SNP. Our model-based genotype calling algorithm captures the redundancy in the system considering all the underlying probe features of a particular SNP, automatically down-weighting any 'bad data' corresponding to image artifacts on the microarray slide or failure of a specific chemistry. In this regard, our method is able to automatically select the probes which work well and reduce the effect of other so-called bad performing probes in a sample-specific manner, for any number of SNPs.

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE PAGES

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; ...

2014-06-01

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Microtiter plate-based antibody microarrays for bacteria and toxins

USDA-ARS?s Scientific Manuscript database

Research has focused on the development of rapid biosensor-based, high-throughput, and multiplexed detection of pathogenic bacteria in foods. Specifically, antibody microarrays in 96-well microtiter plates have been generated for the purpose of selective detection of Shiga toxin-producing E. coli (...

Signal amplification by rolling circle amplification on DNA microarrays

PubMed Central

Nallur, Girish; Luo, Chenghua; Fang, Linhua; Cooley, Stephanie; Dave, Varshal; Lambert, Jeremy; Kukanskis, Kari; Kingsmore, Stephen; Lasken, Roger; Schweitzer, Barry

2001-01-01

While microarrays hold considerable promise in large-scale biology on account of their massively parallel analytical nature, there is a need for compatible signal amplification procedures to increase sensitivity without loss of multiplexing. Rolling circle amplification (RCA) is a molecular amplification method with the unique property of product localization. This report describes the application of RCA signal amplification for multiplexed, direct detection and quantitation of nucleic acid targets on planar glass and gel-coated microarrays. As few as 150 molecules bound to the surface of microarrays can be detected using RCA. Because of the linear kinetics of RCA, nucleic acid target molecules may be measured with a dynamic range of four orders of magnitude. Consequently, RCA is a promising technology for the direct measurement of nucleic acids on microarrays without the need for a potentially biasing preamplification step. PMID:11726701

Exploring homogeneity of correlation structures of gene expression datasets within and between etiological disease categories.

PubMed

Jong, Victor L; Novianti, Putri W; Roes, Kit C B; Eijkemans, Marinus J C

2014-12-01

The literature shows that classifiers perform differently across datasets and that correlations within datasets affect the performance of classifiers. The question that arises is whether the correlation structure within datasets differ significantly across diseases. In this study, we evaluated the homogeneity of correlation structures within and between datasets of six etiological disease categories; inflammatory, immune, infectious, degenerative, hereditary and acute myeloid leukemia (AML). We also assessed the effect of filtering; detection call and variance filtering on correlation structures. We downloaded microarray datasets from ArrayExpress for experiments meeting predefined criteria and ended up with 12 datasets for non-cancerous diseases and six for AML. The datasets were preprocessed by a common procedure incorporating platform-specific recommendations and the two filtering methods mentioned above. Homogeneity of correlation matrices between and within datasets of etiological diseases was assessed using the Box's M statistic on permuted samples. We found that correlation structures significantly differ between datasets of the same and/or different etiological disease categories and that variance filtering eliminates more uncorrelated probesets than detection call filtering and thus renders the data highly correlated.

Challenges of microarray applications for microbial detection and gene expression profiling in food

USDA-ARS?s Scientific Manuscript database

Microarray technology represents one of the latest advances in molecular biology. The diverse types of microarrays have been applied to clinical and environmental microbiology, microbial ecology, and in human, veterinary, and plant diagnostics. Since multiple genes can be analyzed simultaneously, ...

Spot detection and image segmentation in DNA microarray data.

PubMed

Qin, Li; Rueda, Luis; Ali, Adnan; Ngom, Alioune

2005-01-01

Following the invention of microarrays in 1994, the development and applications of this technology have grown exponentially. The numerous applications of microarray technology include clinical diagnosis and treatment, drug design and discovery, tumour detection, and environmental health research. One of the key issues in the experimental approaches utilising microarrays is to extract quantitative information from the spots, which represent genes in a given experiment. For this process, the initial stages are important and they influence future steps in the analysis. Identifying the spots and separating the background from the foreground is a fundamental problem in DNA microarray data analysis. In this review, we present an overview of state-of-the-art methods for microarray image segmentation. We discuss the foundations of the circle-shaped approach, adaptive shape segmentation, histogram-based methods and the recently introduced clustering-based techniques. We analytically show that clustering-based techniques are equivalent to the one-dimensional, standard k-means clustering algorithm that utilises the Euclidean distance.

Women's experiences receiving abnormal prenatal chromosomal microarray testing results.

PubMed

Bernhardt, Barbara A; Soucier, Danielle; Hanson, Karen; Savage, Melissa S; Jackson, Laird; Wapner, Ronald J

2013-02-01

Genomic microarrays can detect copy-number variants not detectable by conventional cytogenetics. This technology is diffusing rapidly into prenatal settings even though the clinical implications of many copy-number variants are currently unknown. We conducted a qualitative pilot study to explore the experiences of women receiving abnormal results from prenatal microarray testing performed in a research setting. Participants were a subset of women participating in a multicenter prospective study "Prenatal Cytogenetic Diagnosis by Array-based Copy Number Analysis." Telephone interviews were conducted with 23 women receiving abnormal prenatal microarray results. We found that five key elements dominated the experiences of women who had received abnormal prenatal microarray results: an offer too good to pass up, blindsided by the results, uncertainty and unquantifiable risks, need for support, and toxic knowledge. As prenatal microarray testing is increasingly used, uncertain findings will be common, resulting in greater need for careful pre- and posttest counseling, and more education of and resources for providers so they can adequately support the women who are undergoing testing.

Emergent FDA biodefense issues for microarray technology: process analytical technology.

PubMed

Weinberg, Sandy

2004-11-01

A successful biodefense strategy relies upon any combination of four approaches. A nation can protect its troops and citizenry first by advanced mass vaccination, second, by responsive ring vaccination, and third, by post-exposure therapeutic treatment (including vaccine therapies). Finally, protection can be achieved by rapid detection followed by exposure limitation (suites and air filters) or immediate treatment (e.g., antibiotics, rapid vaccines and iodine pills). All of these strategies rely upon or are enhanced by microarray technologies. Microarrays can be used to screen, engineer and test vaccines. They are also used to construct early detection tools. While effective biodefense utilizes a variety of tactical tools, microarray technology is a valuable arrow in that quiver.

Biological relevance of CNV calling methods using familial relatedness including monozygotic twins.

PubMed

Castellani, Christina A; Melka, Melkaye G; Wishart, Andrea E; Locke, M Elizabeth O; Awamleh, Zain; O'Reilly, Richard L; Singh, Shiva M

2014-04-21

Studies involving the analysis of structural variation including Copy Number Variation (CNV) have recently exploded in the literature. Furthermore, CNVs have been associated with a number of complex diseases and neurodevelopmental disorders. Common methods for CNV detection use SNP, CNV, or CGH arrays, where the signal intensities of consecutive probes are used to define the number of copies associated with a given genomic region. These practices pose a number of challenges that interfere with the ability of available methods to accurately call CNVs. It has, therefore, become necessary to develop experimental protocols to test the reliability of CNV calling methods from microarray data so that researchers can properly discriminate biologically relevant data from noise. We have developed a workflow for the integration of data from multiple CNV calling algorithms using the same array results. It uses four CNV calling programs: PennCNV (PC), Affymetrix® Genotyping Console™ (AGC), Partek® Genomics Suite™ (PGS) and Golden Helix SVS™ (GH) to analyze CEL files from the Affymetrix® Human SNP 6.0 Array™. To assess the relative suitability of each program, we used individuals of known genetic relationships. We found significant differences in CNV calls obtained by different CNV calling programs. Although the programs showed variable patterns of CNVs in the same individuals, their distribution in individuals of different degrees of genetic relatedness has allowed us to offer two suggestions. The first involves the use of multiple algorithms for the detection of the largest possible number of CNVs, and the second suggests the use of PennCNV over all other methods when the use of only one software program is desirable.

Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB-Based Oligonucleotide Microarray

PubMed Central

Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun

2014-01-01

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes. PMID:24185846

Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

PubMed

Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun; Cha, Hyung Joon

2014-01-01

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

DNA Microarray for Detection of Gastrointestinal Viruses

PubMed Central

Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

2014-01-01

Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help in understanding the etiology of gastroenteritis in humans and animals. PMID:25355758

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminatedmore » the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.« less

Nanotechnology: moving from microarrays toward nanoarrays.

PubMed

Chen, Hua; Li, Jun

2007-01-01

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

[Typing and subtyping avian influenza virus using DNA microarrays].

PubMed

Yang, Zhongping; Wang, Xiurong; Tian, Lina; Wang, Yu; Chen, Hualan

2008-07-01

Outbreaks of highly pathogenic avian influenza (HPAI) virus has caused great economic loss to the poultry industry and resulted in human deaths in Thailand and Vietnam since 2004. Rapid typing and subtyping of viruses, especially HPAI from clinical specimens, are desirable for taking prompt control measures to prevent spreading of the disease. We described a simultaneous approach using microarray to detect and subtype avian influenza virus (AIV). We designed primers of probe genes and used reverse transcriptase PCR to prepare cDNAs of AIV M gene, H5, H7, H9 subtypes haemagglutinin genes and N1, N2 subtypes neuraminidase genes. They were cloned, sequenced, reamplified and spotted to form a glass-bound microarrays. We labeled samples using Cy3-dUTP by RT-PCR, hybridized and scanned the microarrays to typing and subtyping AIV. The hybridization pattern agreed perfectly with the known grid location of each probe, no cross hybridization could be detected. Examinating of HA subtypes 1 through 15, 30 infected samples and 21 field samples revealed the DNA microarray assay was more sensitive and specific than RT-PCR test and chicken embryo inoculation. It can simultaneously detect and differentiate the main epidemic AIV. The results show that DNA microarray technology is a useful diagnostic method.

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics▿

PubMed Central

Zhao, Zhengshan; Peytavi, Régis; Diaz-Quijada, Gerardo A.; Picard, Francois J.; Huletsky, Ann; Leblanc, Éric; Frenette, Johanne; Boivin, Guy; Veres, Teodor; Dumoulin, Michel M.; Bergeron, Michel G.

2008-01-01

Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations. PMID:18784318

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri J.; Stam, Christina N.; Andersen, Gary L.; DeSantis, Todd

2011-01-01

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Variance stabilization and normalization for one-color microarray data using a data-driven multiscale approach.

PubMed

Motakis, E S; Nason, G P; Fryzlewicz, P; Rutter, G A

2006-10-15

Many standard statistical techniques are effective on data that are normally distributed with constant variance. Microarray data typically violate these assumptions since they come from non-Gaussian distributions with a non-trivial mean-variance relationship. Several methods have been proposed that transform microarray data to stabilize variance and draw its distribution towards the Gaussian. Some methods, such as log or generalized log, rely on an underlying model for the data. Others, such as the spread-versus-level plot, do not. We propose an alternative data-driven multiscale approach, called the Data-Driven Haar-Fisz for microarrays (DDHFm) with replicates. DDHFm has the advantage of being 'distribution-free' in the sense that no parametric model for the underlying microarray data is required to be specified or estimated; hence, DDHFm can be applied very generally, not just to microarray data. DDHFm achieves very good variance stabilization of microarray data with replicates and produces transformed intensities that are approximately normally distributed. Simulation studies show that it performs better than other existing methods. Application of DDHFm to real one-color cDNA data validates these results. The R package of the Data-Driven Haar-Fisz transform (DDHFm) for microarrays is available in Bioconductor and CRAN.

Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

PubMed Central

Biyani, Manish; Ichiki, Takanori

2015-01-01

Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA)-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing) a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density), ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era. PMID:27600226

Development of a cell microarray chip for detection of circulating tumor cells

NASA Astrophysics Data System (ADS)

Yamamura, S.; Yatsushiro, S.; Abe, K.; Baba, Y.; Kataoka, M.

2012-03-01

Detection of circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients has clinical significance in earlier diagnosis of metastases. In this study, a novel cell microarray chip for accurate and rapid detection of tumor cells from human leukocytes was developed. The chip with 20,944 microchambers (105 μm diameter and 50 μm depth) was made from polystyrene, and the surface was rendered to hydrophilic by means of reactive-ion etching, which led to the formation of mono-layers of leukocytes on the microchambers. As the model of CTCs detection, we spiked human bronchioalveolar carcinoma (H1650) cells into human T lymphoblastoid leukemia (CEM) cells suspension and detected H1650 cells using the chip. A CEM suspension contained with H1650 cells was dispersed on the chip surface, followed by 10 min standing to allow the cells to settle down into the microchambers. About 30 CEM cells were accommodated in each microchamber, over 600,000 CEM cells in total being on a chip. We could detect 1 H1650 cell per 106 CEM cells on the microarray by staining with fluorescence-conjugated antibody (Anti-Cytokeratin) and cell membrane marker (DiD). Thus, this cell microarray chip has highly potential to be a novel tool of accurate and rapid detection of CTCs.

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

USDA-ARS?s Scientific Manuscript database

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal and polyclonal anti...

Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

PubMed

Li, Yongjin

2016-01-01

The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use.

Universal ligation-detection-reaction microarray applied for compost microbes

PubMed Central

Hultman, Jenni; Ritari, Jarmo; Romantschuk, Martin; Paulin, Lars; Auvinen, Petri

2008-01-01

Background Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR) based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones. Results Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS) area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array. Conclusion This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities. PMID:19116002

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

PubMed Central

Muller, Jean; Mehlen, André; Vetter, Guillaume; Yatskou, Mikalai; Muller, Arnaud; Chalmel, Frédéric; Poch, Olivier; Friederich, Evelyne; Vallar, Laurent

2007-01-01

Background The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton. Results Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not. Conclusion Our data demonstrate that Actichip is a powerful alternative to commercial high density microarrays for cytoskeleton gene profiling in normal or pathological samples. Actichip is available upon request. PMID:17727702

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice.

PubMed

Bruno, D L; Ganesamoorthy, D; Schoumans, J; Bankier, A; Coman, D; Delatycki, M; Gardner, R J M; Hunter, M; James, P A; Kannu, P; McGillivray, G; Pachter, N; Peters, H; Rieubland, C; Savarirayan, R; Scheffer, I E; Sheffield, L; Tan, T; White, S M; Yeung, A; Bowman, Z; Ngo, C; Choy, K W; Cacheux, V; Wong, L; Amor, D J; Slater, H R

2009-02-01

Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

A label-free, fluorescence based assay for microarray

NASA Astrophysics Data System (ADS)

Niu, Sanjun

DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same intensity of excitation light. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested.

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

PubMed

Kim, Chang Sup; Seo, Jeong Hyun; Cha, Hyung Joon

2012-08-07

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

PubMed Central

Scheler, Ott; Glynn, Barry; Parkel, Sven; Palta, Priit; Toome, Kadri; Kaplinski, Lauris; Remm, Maido; Maher, Majella; Kurg, Ants

2009-01-01

Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology. PMID:19445684

Direct Detection of Drug-Resistant Hepatitis B Virus in Serum Using a Dendron-Modified Microarray

PubMed Central

Kim, Doo Hyun; Kang, Hong Seok; Hur, Seong-Suk; Sim, Seobo; Ahn, Sung Hyun; Park, Yong Kwang; Park, Eun-Sook; Lee, Ah Ram; Park, Soree; Kwon, So Young; Lee, Jeong-Hoon

2018-01-01

Background/Aims Direct sequencing is the gold standard for the detection of drug-resistance mutations in hepatitis B virus (HBV); however, this procedure is time-consuming, labor-intensive, and difficult to adapt to high-throughput screening. In this study, we aimed to develop a dendron-modified DNA microarray for the detection of genotypic resistance mutations and evaluate its efficiency. Methods The specificity, sensitivity, and selectivity of dendron-modified slides for the detection of representative drug-resistance mutations were evaluated and compared to those of conventional slides. The diagnostic accuracy was validated using sera obtained from 13 patients who developed viral breakthrough during lamivudine, adefovir, or entecavir therapy and compared with the accuracy of restriction fragment mass polymorphism and direct sequencing data. Results The dendron-modified slides significantly outperformed the conventional microarray slides and were able to detect HBV DNA at a very low level (1 copy/μL). Notably, HBV mutants could be detected in the chronic hepatitis B patient sera without virus purification. The validation of our data revealed that this technique is fully compatible with sequencing data of drug-resistant HBV. Conclusions We developed a novel diagnostic technique for the simultaneous detection of several drug-resistance mutations using a dendron-modified DNA microarray. This technique can be directly applied to sera from chronic hepatitis B patients who show resistance to several nucleos(t)ide analogues. PMID:29271185

High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples.

PubMed

Gallo Vaulet, Lucía; Entrocassi, Carolina; Portu, Ana I; Castro, Erica; Di Bartolomeo, Susana; Ruettger, Anke; Sachse, Konrad; Rodriguez Fermepin, Marcelo

2016-01-01

Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F.

A novel piezoelectric quartz micro-array immunosensor for detection of immunoglobulinE.

PubMed

Yao, Chunyan; Chen, Qinghai; Chen, Ming; Zhang, Bo; Luo, Yang; Huang, Qing; Huang, Junfu; Fu, Weiling

2006-12-01

A novel multi-channel 2 x 5 model of piezoelectric (PZ) micro-array immunosensor has been developed for quantitative detection of human immunoglobulinE (IgE) in serum. Every crystal unit of the fabricated piezoelectric IgE micro-array immunosensor can oscillate without interfering each other. A multi-channel 2 x 5 model micro-array immunosensor as compared with the traditional one-channel immunosensor can provide eight times higher detection speeds for IgE assay. The anti-IgE antibody is deposited on the gold electrode's surface of 10 MHz AT-cut quartz crystals by SPA (staphylococcal protein A), and serves as an antibody recognizing layer. The highly ordered antibody monolayers ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The uniform amount of antibody monolayer coated by the SPA is good, and non-specific reaction caused by other immunoglobulin in sample is found. The fabricated PZ immunosensor can be used for human IgE determination in the range of 5-300 IU/ml with high precision (CV is 4%). 50 human serum samples were detected by the micro-array immunosensor, and the results agreed well with those given by the commercially ELISA test kits. The correlation coefficient is 0.94 between ELISA and PZ immunosensor. After regeneration with NaOH the coated immunosensor can be reused 6 times without appreciable loss of activity.

Automatic image analysis and spot classification for detection of pathogenic Escherichia coli on glass slide DNA microarrays

USDA-ARS?s Scientific Manuscript database

A computer algorithm was created to inspect scanned images from DNA microarray slides developed to rapidly detect and genotype E. Coli O157 virulent strains. The algorithm computes centroid locations for signal and background pixels in RGB space and defines a plane perpendicular to the line connect...

Crossword: A Fully Automated Algorithm for the Segmentation and Quality Control of Protein Microarray Images

PubMed Central

2015-01-01

Biological assays formatted as microarrays have become a critical tool for the generation of the comprehensive data sets required for systems-level understanding of biological processes. Manual annotation of data extracted from images of microarrays, however, remains a significant bottleneck, particularly for protein microarrays due to the sensitivity of this technology to weak artifact signal. In order to automate the extraction and curation of data from protein microarrays, we describe an algorithm called Crossword that logically combines information from multiple approaches to fully automate microarray segmentation. Automated artifact removal is also accomplished by segregating structured pixels from the background noise using iterative clustering and pixel connectivity. Correlation of the location of structured pixels across image channels is used to identify and remove artifact pixels from the image prior to data extraction. This component improves the accuracy of data sets while reducing the requirement for time-consuming visual inspection of the data. Crossword enables a fully automated protocol that is robust to significant spatial and intensity aberrations. Overall, the average amount of user intervention is reduced by an order of magnitude and the data quality is increased through artifact removal and reduced user variability. The increase in throughput should aid the further implementation of microarray technologies in clinical studies. PMID:24417579

The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

PubMed Central

Erlandsson, Lena; Rosenstierne, Maiken W.; McLoughlin, Kevin; Jaing, Crystal; Fomsgaard, Anders

2011-01-01

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. PMID:21853040

DNA microarrays: a powerful genomic tool for biomedical and clinical research

PubMed Central

Trevino, Victor; Falciani, Francesco; Barrera-Saldaña, Hugo A.

2007-01-01

Among the many benefits of the Human Genome Project are new and powerful tools such as the genome-wide hybridization devices referred as microarrays. Initially designed to measure gene transcriptional levels, microarray technologies are now used for comparing other genome features among individuals and their tissues and cells. Results provide valuable information on disease subcategories, disease prognosis, and treatment outcome. Likewise, reveal differences in genetic makeup, regulatory mechanisms and subtle variations are approaching the era of personalized medicine. To understand this powerful tool, its versatility and how it is dramatically changing the molecular approach to biomedical and clinical research, this review describes the technology, its applications, a didactic step-by-step review of a typical microarray protocol, and a real experiment. Finally, it calls the attention of the medical community to integrate multidisciplinary teams, to take advantage of this technology and its expanding applications that in a slide reveals our genetic inheritance and destiny. PMID:17660860

A Java-based tool for the design of classification microarrays.

PubMed

Meng, Da; Broschat, Shira L; Call, Douglas R

2008-08-04

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for analysis of subsequent experimental data. Additionally, PLASMID can be used to construct virtual microarrays with genomes from public databases, which can then be used to identify an optimal set of probes.

Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization.

PubMed

Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak

2016-04-05

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

PubMed

Ma, Y; Dai, X; Hong, T; Munk, G B; Libera, M

2016-12-19

Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 μm × 250 μm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 μm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

NASA Astrophysics Data System (ADS)

Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

2011-06-01

Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

Rapid Spoligotyping of Mycobacterium tuberculosis Complex Bacteria by Use of a Microarray System with Automatic Data Processing and Assignment

PubMed Central

Ruettger, Anke; Nieter, Johanna; Skrypnyk, Artem; Engelmann, Ines; Ziegler, Albrecht; Moser, Irmgard; Monecke, Stefan; Ehricht, Ralf

2012-01-01

Membrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assay's validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability. PMID:22553239

Rapid spoligotyping of Mycobacterium tuberculosis complex bacteria by use of a microarray system with automatic data processing and assignment.

PubMed

Ruettger, Anke; Nieter, Johanna; Skrypnyk, Artem; Engelmann, Ines; Ziegler, Albrecht; Moser, Irmgard; Monecke, Stefan; Ehricht, Ralf; Sachse, Konrad

2012-07-01

Membrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assay's validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability.

Applications of microarray technology in breast cancer research

PubMed Central

Cooper, Colin S

2001-01-01

Microarrays provide a versatile platform for utilizing information from the Human Genome Project to benefit human health. This article reviews the ways in which microarray technology may be used in breast cancer research. Its diverse applications include monitoring chromosome gains and losses, tumour classification, drug discovery and development, DNA resequencing, mutation detection and investigating the mechanism of tumour development. PMID:11305951

Creation of antifouling microarrays by photopolymerization of zwitterionic compounds for protein assay and cell patterning.

PubMed

Sun, Xiuhua; Wang, Huaixin; Wang, Yuanyuan; Gui, Taijiang; Wang, Ke; Gao, Changlu

2018-04-15

Nonspecific binding or adsorption of biomolecules presents as a major obstacle to higher sensitivity, specificity and reproducibility in microarray technology. We report herein a method to fabricate antifouling microarray via photopolymerization of biomimetic betaine compounds. In brief, carboxybetaine methacrylate was polymerized as arrays for protein sensing, while sulfobetaine methacrylate was polymerized as background. With the abundant carboxyl groups on array surfaces and zwitterionic polymers on the entire surfaces, this microarray allows biomolecular immobilization and recognition with low nonspecific interactions due to its antifouling property. Therefore, low concentration of target molecules can be captured and detected by this microarray. It was proved that a concentration of 10ngmL -1 bovine serum albumin in the sample matrix of bovine serum can be detected by the microarray derivatized with anti-bovine serum albumin. Moreover, with proper hydrophilic-hydrophobic designs, this approach can be applied to fabricate surface-tension droplet arrays, which allows surface-directed cell adhesion and growth. These light controllable approaches constitute a clear improvement in the design of antifouling interfaces, which may lead to greater flexibility in the development of interfacial architectures and wider application in blood contact microdevices. Copyright © 2017 Elsevier B.V. All rights reserved.

Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2000-12-01

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2001-07-05

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Apparently low reproducibility of true differential expression discoveries in microarray studies.

PubMed

Zhang, Min; Yao, Chen; Guo, Zheng; Zou, Jinfeng; Zhang, Lin; Xiao, Hui; Wang, Dong; Yang, Da; Gong, Xue; Zhu, Jing; Li, Yanhui; Li, Xia

2008-09-15

Differentially expressed gene (DEG) lists detected from different microarray studies for a same disease are often highly inconsistent. Even in technical replicate tests using identical samples, DEG detection still shows very low reproducibility. It is often believed that current small microarray studies will largely introduce false discoveries. Based on a statistical model, we show that even in technical replicate tests using identical samples, it is highly likely that the selected DEG lists will be very inconsistent in the presence of small measurement variations. Therefore, the apparently low reproducibility of DEG detection from current technical replicate tests does not indicate low quality of microarray technology. We also demonstrate that heterogeneous biological variations existing in real cancer data will further reduce the overall reproducibility of DEG detection. Nevertheless, in small subsamples from both simulated and real data, the actual false discovery rate (FDR) for each DEG list tends to be low, suggesting that each separately determined list may comprise mostly true DEGs. Rather than simply counting the overlaps of the discovery lists from different studies for a complex disease, novel metrics are needed for evaluating the reproducibility of discoveries characterized with correlated molecular changes. Supplementaty information: Supplementary data are available at Bioinformatics online.

Differential gene expression detection and sample classification using penalized linear regression models.

PubMed

Wu, Baolin

2006-02-15

Differential gene expression detection and sample classification using microarray data have received much research interest recently. Owing to the large number of genes p and small number of samples n (p > n), microarray data analysis poses big challenges for statistical analysis. An obvious problem owing to the 'large p small n' is over-fitting. Just by chance, we are likely to find some non-differentially expressed genes that can classify the samples very well. The idea of shrinkage is to regularize the model parameters to reduce the effects of noise and produce reliable inferences. Shrinkage has been successfully applied in the microarray data analysis. The SAM statistics proposed by Tusher et al. and the 'nearest shrunken centroid' proposed by Tibshirani et al. are ad hoc shrinkage methods. Both methods are simple, intuitive and prove to be useful in empirical studies. Recently Wu proposed the penalized t/F-statistics with shrinkage by formally using the (1) penalized linear regression models for two-class microarray data, showing good performance. In this paper we systematically discussed the use of penalized regression models for analyzing microarray data. We generalize the two-class penalized t/F-statistics proposed by Wu to multi-class microarray data. We formally derive the ad hoc shrunken centroid used by Tibshirani et al. using the (1) penalized regression models. And we show that the penalized linear regression models provide a rigorous and unified statistical framework for sample classification and differential gene expression detection.

Resequencing Pathogen Microarray (RPM) for prospective detection and identification of emergent pathogen strains and variants

NASA Astrophysics Data System (ADS)

Tibbetts, Clark; Lichanska, Agnieszka M.; Borsuk, Lisa A.; Weslowski, Brian; Morris, Leah M.; Lorence, Matthew C.; Schafer, Klaus O.; Campos, Joseph; Sene, Mohamadou; Myers, Christopher A.; Faix, Dennis; Blair, Patrick J.; Brown, Jason; Metzgar, David

2010-04-01

High-density resequencing microarrays support simultaneous detection and identification of multiple viral and bacterial pathogens. Because detection and identification using RPM is based upon multiple specimen-specific target pathogen gene sequences generated in the individual test, the test results enable both a differential diagnostic analysis and epidemiological tracking of detected pathogen strains and variants from one specimen to the next. The RPM assay enables detection and identification of pathogen sequences that share as little as 80% sequence similarity to prototype target gene sequences represented as detector tiles on the array. This capability enables the RPM to detect and identify previously unknown strains and variants of a detected pathogen, as in sentinel cases associated with an infectious disease outbreak. We illustrate this capability using assay results from testing influenza A virus vaccines configured with strains that were first defined years after the design of the RPM microarray. Results are also presented from RPM-Flu testing of three specimens independently confirmed to the positive for the 2009 Novel H1N1 outbreak strain of influenza virus.

Chemiluminescence microarrays in analytical chemistry: a critical review.

PubMed

Seidel, Michael; Niessner, Reinhard

2014-09-01

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

PubMed

Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

2011-06-01

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

A Customized DNA Microarray for Microbial Source Tracking ...

EPA Pesticide Factsheets

It is estimated that more than 160, 000 miles of rivers and streams in the United States are impaired due to the presence of waterborne pathogens. These pathogens typically originate from human and other animal fecal pollution sources; therefore, a rapid microbial source tracking (MST) method is needed to facilitate water quality assessment and impaired water remediation. We report a novel qualitative DNA microarray technology consisting of 453 probes for the detection of general fecal and host-associated bacteria, viruses, antibiotic resistance, and other environmentally relevant genetic indicators. A novel data normalization and reduction approach is also presented to help alleviate false positives often associated with high-density microarray applications. To evaluate the performance of the approach, DNA and cDNA was isolated from swine, cattle, duck, goose and gull fecal reference samples, as well as soiled poultry liter and raw municipal sewage. Based on nonmetric multidimensional scaling analysis of results, findings suggest that the novel microarray approach may be useful for pathogen detection and identification of fecal contamination in recreational waters. The ability to simultaneously detect a large collection of environmentally important genetic indicators in a single test has the potential to provide water quality managers with a wide range of information in a short period of time. Future research is warranted to measure microarray performance i

Microbial Diagnostic Microarrays for the Detection and Typing of Food- and Water-Borne (Bacterial) Pathogens

PubMed Central

Kostić, Tanja; Sessitsch, Angela

2011-01-01

Reliable and sensitive pathogen detection in clinical and environmental (including food and water) samples is of greatest importance for public health. Standard microbiological methods have several limitations and improved alternatives are needed. Most important requirements for reliable analysis include: (i) specificity; (ii) sensitivity; (iii) multiplexing potential; (iv) robustness; (v) speed; (vi) automation potential; and (vii) low cost. Microarray technology can, through its very nature, fulfill many of these requirements directly and the remaining challenges have been tackled. In this review, we attempt to compare performance characteristics of the microbial diagnostic microarrays developed for the detection and typing of food and water pathogens, and discuss limitations, points still to be addressed and issues specific for the analysis of food, water and environmental samples. PMID:27605332

Exploiting fluorescence for multiplex immunoassays on protein microarrays

NASA Astrophysics Data System (ADS)

Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

2014-09-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Evaluation of two outlier-detection-based methods for detecting tissue-selective genes from microarray data.

PubMed

Kadota, Koji; Konishi, Tomokazu; Shimizu, Kentaro

2007-05-01

Large-scale expression profiling using DNA microarrays enables identification of tissue-selective genes for which expression is considerably higher and/or lower in some tissues than in others. Among numerous possible methods, only two outlier-detection-based methods (an AIC-based method and Sprent's non-parametric method) can treat equally various types of selective patterns, but they produce substantially different results. We investigated the performance of these two methods for different parameter settings and for a reduced number of samples. We focused on their ability to detect selective expression patterns robustly. We applied them to public microarray data collected from 36 normal human tissue samples and analyzed the effects of both changing the parameter settings and reducing the number of samples. The AIC-based method was more robust in both cases. The findings confirm that the use of the AIC-based method in the recently proposed ROKU method for detecting tissue-selective expression patterns is correct and that Sprent's method is not suitable for ROKU.

New diagnostics for melanoma detection: from artificial intelligence to RNA microarrays.

PubMed

Ahlgrimm-Siess, Verena; Laimer, Martin; Arzberger, Edith; Hofmann-Wellenhof, Rainer

2012-07-01

Early detection of melanoma remains crucial to ensuring a favorable prognosis. Dermoscopy and total body photography are well-established noninvasive aids that increase the diagnostic accuracy of dermatologists in their daily routine, beyond that of a naked-eye examination. New noninvasive diagnostic techniques, such as reflectance confocal microscopy, multispectral digital imaging and RNA microarrays, are currently being investigated to determine their utility for melanoma detection. This review presents emerging technologies for noninvasive melanoma diagnosis, and discusses their advantages and limitations.

High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

PubMed

Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

2015-01-01

Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

Geiger mode avalanche photodiodes for microarray systems

NASA Astrophysics Data System (ADS)

Phelan, Don; Jackson, Carl; Redfern, R. Michael; Morrison, Alan P.; Mathewson, Alan

2002-06-01

New Geiger Mode Avalanche Photodiodes (GM-APD) have been designed and characterized specifically for use in microarray systems. Critical parameters such as excess reverse bias voltage, hold-off time and optimum operating temperature have been experimentally determined for these photon-counting devices. The photon detection probability, dark count rate and afterpulsing probability have been measured under different operating conditions. An active- quench circuit (AQC) is presented for operating these GM- APDs. This circuit is relatively simple, robust and has such benefits as reducing average power dissipation and afterpulsing. Arrays of these GM-APDs have already been designed and together with AQCs open up the possibility of having a solid-state microarray detector that enables parallel analysis on a single chip. Another advantage of these GM-APDs over current technology is their low voltage CMOS compatibility which could allow for the fabrication of an AQC on the same device. Small are detectors have already been employed in the time-resolved detection of fluorescence from labeled proteins. It is envisaged that operating these new GM-APDs with this active-quench circuit will have numerous applications for the detection of fluorescence in microarray systems.

Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S; Jaing, C

2012-03-27

The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interimmore » report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.« less

Impact of genotyping errors on statistical power of association tests in genomic analyses: A case study

PubMed Central

Hou, Lin; Sun, Ning; Mane, Shrikant; Sayward, Fred; Rajeevan, Nallakkandi; Cheung, Kei-Hoi; Cho, Kelly; Pyarajan, Saiju; Aslan, Mihaela; Miller, Perry; Harvey, Philip D.; Gaziano, J. Michael; Concato, John; Zhao, Hongyu

2017-01-01

A key step in genomic studies is to assess high throughput measurements across millions of markers for each participant’s DNA, either using microarrays or sequencing techniques. Accurate genotype calling is essential for downstream statistical analysis of genotype-phenotype associations, and next generation sequencing (NGS) has recently become a more common approach in genomic studies. How the accuracy of variant calling in NGS-based studies affects downstream association analysis has not, however, been studied using empirical data in which both microarrays and NGS were available. In this article, we investigate the impact of variant calling errors on the statistical power to identify associations between single nucleotides and disease, and on associations between multiple rare variants and disease. Both differential and nondifferential genotyping errors are considered. Our results show that the power of burden tests for rare variants is strongly influenced by the specificity in variant calling, but is rather robust with regard to sensitivity. By using the variant calling accuracies estimated from a substudy of a Cooperative Studies Program project conducted by the Department of Veterans Affairs, we show that the power of association tests is mostly retained with commonly adopted variant calling pipelines. An R package, GWAS.PC, is provided to accommodate power analysis that takes account of genotyping errors (http://zhaocenter.org/software/). PMID:28019059

Protein microarray with horseradish peroxidase chemiluminescence for quantification of serum α-fetoprotein.

PubMed

Zhao, Yuanshun; Zhang, Yonghong; Lin, Dongdong; Li, Kang; Yin, Chengzeng; Liu, Xiuhong; Jin, Boxun; Sun, Libo; Liu, Jinhua; Zhang, Aiying; Li, Ning

2015-10-01

To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of α-fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC). A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA). Serum AFP concentrations determined via protein microarray were positively correlated (r = 0.973) with those determined via ELISA in patients with HCC (n = 60) and healthy control subjects (n = 30). Protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. Protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974; 95% CI 0.000, 1.000). Protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum. © The Author(s) 2015.

Microfluidic extraction and microarray detection of biomarkers from cancer tissue slides

NASA Astrophysics Data System (ADS)

Nguyen, H. T.; Dupont, L. N.; Jean, A. M.; Géhin, T.; Chevolot, Y.; Laurenceau, E.; Gijs, M. A. M.

2018-03-01

We report here a new microfluidic method allowing for the quantification of human epidermal growth factor receptor 2 (HER2) expression levels from formalin-fixed breast cancer tissues. After partial extraction of proteins from the tissue slide, the extract is routed to an antibody (Ab) microarray for HER2 titration by fluorescence. Then the HER2-expressing cell area is evaluated by immunofluorescence (IF) staining of the tissue slide and used to normalize the fluorescent HER2 signal measured from the Ab microarray. The number of HER2 gene copies measured by fluorescence in situ hybridization (FISH) on an adjacent tissue slide is concordant with the normalized HER2 expression signal. This work is the first study implementing biomarker extraction and detection from cancer tissue slides using microfluidics in combination with a microarray system, paving the way for further developments towards multiplex and precise quantification of cancer biomarkers.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

PubMed

Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

2015-01-01

Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays

PubMed Central

Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

2012-01-01

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. PMID:22772038

Improved microarray methods for profiling the yeast knockout strain collection

PubMed Central

Yuan, Daniel S.; Pan, Xuewen; Ooi, Siew Loon; Peyser, Brian D.; Spencer, Forrest A.; Irizarry, Rafael A.; Boeke, Jef D.

2005-01-01

A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens. PMID:15994458

Chromosomal Microarray Analysis of Consecutive Individuals with Autism Spectrum Disorders Using an Ultra-High Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

PubMed

Ho, Karen S; Wassman, E Robert; Baxter, Adrianne L; Hensel, Charles H; Martin, Megan M; Prasad, Aparna; Twede, Hope; Vanzo, Rena J; Butler, Merlin G

2016-12-09

Copy number variants (CNVs) detected by chromosomal microarray analysis (CMA) significantly contribute to understanding the etiology of autism spectrum disorder (ASD) and other related conditions. In recognition of the value of CMA testing and its impact on medical management, CMA is in medical guidelines as a first-tier test in the evaluation of children with these disorders. As CMA becomes adopted into routine care for these patients, it becomes increasingly important to report these clinical findings. This study summarizes the results of over 4 years of CMA testing by a CLIA-certified clinical testing laboratory. Using a 2.8 million probe microarray optimized for the detection of CNVs associated with neurodevelopmental disorders, we report an overall CNV detection rate of 28.1% in 10,351 consecutive patients, which rises to nearly 33% in cases without ASD, with only developmental delay/intellectual disability (DD/ID) and/or multiple congenital anomalies (MCA). The overall detection rate for individuals with ASD is also significant at 24.4%. The detection rate and pathogenic yield of CMA vary significantly with the indications for testing, age, and gender, as well as the specialty of the ordering doctor. We note discrete differences in the most common recurrent CNVs found in individuals with or without a diagnosis of ASD.

Automated Immunomagnetic Separation and Microarray Detection of E. coli O157:H7 from Poultry Carcass Rinse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandler, Darrell P.; Brown, Jeremy D.; Call, Douglas R.

2001-09-01

We describe the development and application of a novel electromagnetic flow cell and fluidics system for automated immunomagnetic separation of E. coli directly from unprocessed poultry carcass rinse, and the biochemical coupling of automated sample preparation with nucleic acid microarrays without cell growth. Highly porous nickel foam was used as a magnetic flux conductor. Up to 32% recovery efficiency of 'total' E. coli was achieved within the automated system with 6 sec contact times and 15 minute protocol (from sample injection through elution), statistically similar to cell recovery efficiencies in > 1 hour 'batch' captures. The electromagnet flow cell allowedmore » complete recovery of 2.8 mm particles directly from unprocessed poultry carcass rinse whereas the batch system did not. O157:H7 cells were reproducibly isolated directly from unprocessed poultry rinse with 39% recovery efficiency at 103 cells ml-1 inoculum. Direct plating of washed beads showed positive recovery of O 157:H7 directly from carcass rinse at an inoculum of 10 cells ml-1. Recovered beads were used for direct PCR amplification and microarray detection, with a process-level detection limit (automated cell concentration through microarray detection) of < 103 cells ml-1 carcass rinse. The fluidic system and analytical approach described here are generally applicable to most microbial detection problems and applications.« less

APPLICATION OF CDNA MICROARRAY TECHNOLOGY TO IN VITRO TOXICOLOGY AND THE SELECTION OF GENES FOR A REAL TIME RT-PCR-BASED SCREEN FOR OXIDATIVE STRESS IN HEP-G2 CELLS

EPA Science Inventory

Large-scale analysis of gene expression using cDNA microarrays promises the
rapid detection of the mode of toxicity for drugs and other chemicals. cDNA
microarrays were used to examine chemically-induced alterations of gene
expression in HepG2 cells exposed to oxidative ...

Controlling false-negative errors in microarray differential expression analysis: a PRIM approach.

PubMed

Cole, Steve W; Galic, Zoran; Zack, Jerome A

2003-09-22

Theoretical considerations suggest that current microarray screening algorithms may fail to detect many true differences in gene expression (Type II analytic errors). We assessed 'false negative' error rates in differential expression analyses by conventional linear statistical models (e.g. t-test), microarray-adapted variants (e.g. SAM, Cyber-T), and a novel strategy based on hold-out cross-validation. The latter approach employs the machine-learning algorithm Patient Rule Induction Method (PRIM) to infer minimum thresholds for reliable change in gene expression from Boolean conjunctions of fold-induction and raw fluorescence measurements. Monte Carlo analyses based on four empirical data sets show that conventional statistical models and their microarray-adapted variants overlook more than 50% of genes showing significant up-regulation. Conjoint PRIM prediction rules recover approximately twice as many differentially expressed transcripts while maintaining strong control over false-positive (Type I) errors. As a result, experimental replication rates increase and total analytic error rates decline. RT-PCR studies confirm that gene inductions detected by PRIM but overlooked by other methods represent true changes in mRNA levels. PRIM-based conjoint inference rules thus represent an improved strategy for high-sensitivity screening of DNA microarrays. Freestanding JAVA application at http://microarray.crump.ucla.edu/focus

Living Cell Microarrays: An Overview of Concepts

PubMed Central

Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

2016-01-01

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

Automated microfluidic assay system for autoantibodies found in autoimmune diseases using a photoimmobilized autoantigen microarray.

PubMed

Matsudaira, Takahiro; Tsuzuki, Saki; Wada, Akira; Suwa, Akira; Kohsaka, Hitoshi; Tomida, Maiko; Ito, Yoshihiro

2008-01-01

Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune diabetes are characterized by the production of autoantibodies that serve as useful diagnostic markers, surrogate markers, and prognostic factors. We devised an in vitro system to detect these clinically pivotal autoantibodies using a photoimmobilized autoantigen microarray. Photoimmobilization was useful for preparing the autoantigen microarray, where autoantigens are covalently immobilized on a plate, because it does not require specific functional groups of the autoantigens and any organic material can be immobilized by a radical reaction induced by photoirradiation. Here, we prepared the microarray using a very convenient method. Aqueous solutions of each autoantigen were mixed with a polymer of poly(ethylene glycol) methacrylate and a photoreactive crosslinker, and the mixtures were microspotted on a plate and dried in air. Finally, the plate was irradiated with an ultraviolet lamp to obtain immobilization. In the assay, patient serum was added to the microarray plate. Antigen-specific IgG adsorbed on the microspotted autoantigen was detected by peroxidase-conjugated anti-IgG antibody. The chemical luminescence intensities of the substrate decomposed by the peroxidase were detected with a sensitive CCD camera. All autoantigens were immobilized stably by this method and used to screen antigen-specific IgG. In addition, the plate was covered with a polydimethylsiloxane sheet containing microchannels and automated measurement was carried out.

Integrated Microfluidic Devices for Automated Microarray-Based Gene Expression and Genotyping Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Lodes, Mike; Fuji, H. Sho; Danley, David; McShea, Andrew

Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray® chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The micro-fluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.

Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

PubMed Central

Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

2010-01-01

Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

Learning a single-hidden layer feedforward neural network using a rank correlation-based strategy with application to high dimensional gene expression and proteomic spectra datasets in cancer detection.

PubMed

Belciug, Smaranda; Gorunescu, Florin

2018-06-08

Methods based on microarrays (MA), mass spectrometry (MS), and machine learning (ML) algorithms have evolved rapidly in recent years, allowing for early detection of several types of cancer. A pitfall of these approaches, however, is the overfitting of data due to large number of attributes and small number of instances -- a phenomenon known as the 'curse of dimensionality'. A potentially fruitful idea to avoid this drawback is to develop algorithms that combine fast computation with a filtering module for the attributes. The goal of this paper is to propose a statistical strategy to initiate the hidden nodes of a single-hidden layer feedforward neural network (SLFN) by using both the knowledge embedded in data and a filtering mechanism for attribute relevance. In order to attest its feasibility, the proposed model has been tested on five publicly available high-dimensional datasets: breast, lung, colon, and ovarian cancer regarding gene expression and proteomic spectra provided by cDNA arrays, DNA microarray, and MS. The novel algorithm, called adaptive SLFN (aSLFN), has been compared with four major classification algorithms: traditional ELM, radial basis function network (RBF), single-hidden layer feedforward neural network trained by backpropagation algorithm (BP-SLFN), and support vector-machine (SVM). Experimental results showed that the classification performance of aSLFN is competitive with the comparison models. Copyright © 2018. Published by Elsevier Inc.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

PubMed

Zhang, Huijie; Oellers, Tobias; Feng, Wenqian; Abdulazim, Tarik; Saw, En Ning; Ludwig, Alfred; Levkin, Pavel A; Plumeré, Nicolas

2017-06-06

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

PubMed

El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

2015-01-30

In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Microarray technology for major chemical contaminants analysis in food: current status and prospects.

PubMed

Zhang, Zhaowei; Li, Peiwu; Hu, Xiaofeng; Zhang, Qi; Ding, Xiaoxia; Zhang, Wen

2012-01-01

Chemical contaminants in food have caused serious health issues in both humans and animals. Microarray technology is an advanced technique suitable for the analysis of chemical contaminates. In particular, immuno-microarray approach is one of the most promising methods for chemical contaminants analysis. The use of microarrays for the analysis of chemical contaminants is the subject of this review. Fabrication strategies and detection methods for chemical contaminants are discussed in detail. Application to the analysis of mycotoxins, biotoxins, pesticide residues, and pharmaceutical residues is also described. Finally, future challenges and opportunities are discussed.

Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping

NASA Technical Reports Server (NTRS)

Royce, Thomas E.; Rozowsky, Joel S.; Bertone, Paul; Samanta, Manoj; Stolc, Viktor; Weissman, Sherman; Snyder, Michael; Gerstein, Mark

2005-01-01

Traditional microarrays use probes complementary to known genes to quantitate the differential gene expression between two or more conditions. Genomic tiling microarray experiments differ in that probes that span a genomic region at regular intervals are used to detect the presence or absence of transcription. This difference means the same sets of biases and the methods for addressing them are unlikely to be relevant to both types of experiment. We introduce the informatics challenges arising in the analysis of tiling microarray experiments as open problems to the scientific community and present initial approaches for the analysis of this nascent technology.

Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs).

PubMed

Turkec, Aydin; Lucas, Stuart J; Karacanli, Burçin; Baykut, Aykut; Yuksel, Hakki

2016-03-01

Detection of GMO material in crop and food samples is the primary step in GMO monitoring and regulation, with the increasing number of GM events in the world market requiring detection solutions with high multiplexing capacity. In this study, we test the suitability of a high-density oligonucleotide microarray platform for direct, quantitative detection of GMOs found in the Turkish feed market. We tested 1830 different 60nt probes designed to cover the GM cassettes from 12 different GM cultivars (3 soya, 9 maize), as well as plant species-specific and contamination controls, and developed a data analysis method aiming to provide maximum throughput and sensitivity. The system was able specifically to identify each cultivar, and in 10/12 cases was sensitive enough to detect GMO DNA at concentrations of ⩽1%. These GMOs could also be quantified using the microarray, as their fluorescence signals increased linearly with GMO concentration. Copyright © 2015 Elsevier Ltd. All rights reserved.

A highly oriented hybrid microarray modified electrode fabricated by a template-free method for ultrasensitive electrochemical DNA recognition

NASA Astrophysics Data System (ADS)

Shi, Lei; Chu, Zhenyu; Dong, Xueliang; Jin, Wanqin; Dempsey, Eithne

2013-10-01

Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors.Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors. Electronic supplementary information (ESI) available: Four-probe method for determining the conductivity of the hybrid crystal (Fig. S1); stability comparisons of the hybrid films (Fig. S2); FESEM images of the hybrid microarray (Fig. S3); electrochemical characterizations of the hybrid films (Fig. S4); DFT simulations (Fig. S5); cross-sectional FESEM image of the hybrid microarray (Fig. S6); regeneration and stability tests of the DNA biosensor (Fig. S7). See DOI: 10.1039/c3nr03097k

Development of Fluorescent Polymerization-based Signal Amplification for Sensitive and Non-enzymatic Biodetection in Antibody Microarrays

PubMed Central

Avens, Heather J.; Bowman, Christopher N.

2009-01-01

Antibody microarrays are a critical tool for proteomics, requiring broad, highly sensitive detection of numerous low abundance biomarkers. Fluorescent polymerization-based amplification (FPBA) is presented as a novel, non-enzymatic signal amplification method that takes advantage of the chain-reaction nature of radical polymerization to achieve a highly amplified fluorescent response. A streptavidin-eosin conjugate localizes eosin photoinitiators for polymerization on the chip where biotinylated target protein is bound. The chip is contacted with acrylamide as a monomer, N-methyldiethanolamine as a coinitiator and yellow/green fluorescent nanoparticles (NPs) which, upon initiation, combine to form a macroscopically visible and highly fluorescent film. The rapid polymerization kinetics and the presence of cross-linker favor entrapment of the fluorescent NPs in the polymer, enabling highly sensitive fluorescent biodetection. This method is demonstrated as being appropriate for antibody microarrays and is compared to detection approaches which utilize streptavidin-FITC (SA-FITC) and streptavidin-labeled yellow/green NPs (SA-NPs). It is found that FPBA is able to detect 0.16 (+/− 0.01) biotin-antibody/µm2 (or 40 zeptomole surface-bound target molecules), while SA-FITC has a limit of detection of 31 (+/− 1) biotin-antibody/µm2 and SA-NPs fail to achieve any significant signal under the conditions evaluated here. Further, FPBA in conjunction with fluorescent stereomicroscopy yields equal or better sensitivity compared to fluorescent detection of SA-eosin using a much more costly microarray scanner. By facilitating highly sensitive detection, FPBA is expected to enable detection of low abundance antigens and also make possible a transition towards less expensive fluorescence detection instrumentation. PMID:19508906

Best practices for hybridization design in two-colour microarray analysis.

PubMed

Knapen, Dries; Vergauwen, Lucia; Laukens, Kris; Blust, Ronny

2009-07-01

Two-colour microarrays are a popular platform of choice in gene expression studies. Because two different samples are hybridized on a single microarray, and several microarrays are usually needed in a given experiment, there are many possible ways to combine samples on different microarrays. The actual combination employed is commonly referred to as the 'hybridization design'. Different types of hybridization designs have been developed, all aimed at optimizing the experimental setup for the detection of differentially expressed genes while coping with technical noise. Here, we first provide an overview of the different classes of hybridization designs, discussing their advantages and limitations, and then we illustrate the current trends in the use of different hybridization design types in contemporary research.

Open-target sparse sensing of biological agents using DNA microarray

PubMed Central

2011-01-01

Background Current biosensors are designed to target and react to specific nucleic acid sequences or structural epitopes. These 'target-specific' platforms require creation of new physical capture reagents when new organisms are targeted. An 'open-target' approach to DNA microarray biosensing is proposed and substantiated using laboratory generated data. The microarray consisted of 12,900 25 bp oligonucleotide capture probes derived from a statistical model trained on randomly selected genomic segments of pathogenic prokaryotic organisms. Open-target detection of organisms was accomplished using a reference library of hybridization patterns for three test organisms whose DNA sequences were not included in the design of the microarray probes. Results A multivariate mathematical model based on the partial least squares regression (PLSR) was developed to detect the presence of three test organisms in mixed samples. When all 12,900 probes were used, the model correctly detected the signature of three test organisms in all mixed samples (mean(R2)) = 0.76, CI = 0.95), with a 6% false positive rate. A sampling algorithm was then developed to sparsely sample the probe space for a minimal number of probes required to capture the hybridization imprints of the test organisms. The PLSR detection model was capable of correctly identifying the presence of the three test organisms in all mixed samples using only 47 probes (mean(R2)) = 0.77, CI = 0.95) with nearly 100% specificity. Conclusions We conceived an 'open-target' approach to biosensing, and hypothesized that a relatively small, non-specifically designed, DNA microarray is capable of identifying the presence of multiple organisms in mixed samples. Coupled with a mathematical model applied to laboratory generated data, and sparse sampling of capture probes, the prototype microarray platform was able to capture the signature of each organism in all mixed samples with high sensitivity and specificity. It was demonstrated that this new approach to biosensing closely follows the principles of sparse sensing. PMID:21801424

Evaluation of Two Outlier-Detection-Based Methods for Detecting Tissue-Selective Genes from Microarray Data

PubMed Central

Kadota, Koji; Konishi, Tomokazu; Shimizu, Kentaro

2007-01-01

Large-scale expression profiling using DNA microarrays enables identification of tissue-selective genes for which expression is considerably higher and/or lower in some tissues than in others. Among numerous possible methods, only two outlier-detection-based methods (an AIC-based method and Sprent’s non-parametric method) can treat equally various types of selective patterns, but they produce substantially different results. We investigated the performance of these two methods for different parameter settings and for a reduced number of samples. We focused on their ability to detect selective expression patterns robustly. We applied them to public microarray data collected from 36 normal human tissue samples and analyzed the effects of both changing the parameter settings and reducing the number of samples. The AIC-based method was more robust in both cases. The findings confirm that the use of the AIC-based method in the recently proposed ROKU method for detecting tissue-selective expression patterns is correct and that Sprent’s method is not suitable for ROKU. PMID:19936074

Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

PubMed Central

Jin, Lian-Qun; Li, Jun-Wen; Wang, Sheng-Qi; Chao, Fu-Huan; Wang, Xin-Wei; Yuan, Zheng-Quan

2005-01-01

AIM: To detect the common intestinal pathogenic bacteria quickly and accurately. METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays. RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified. CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost. PMID:16437687

Recognition of multiple imbalanced cancer types based on DNA microarray data using ensemble classifiers.

PubMed

Yu, Hualong; Hong, Shufang; Yang, Xibei; Ni, Jun; Dan, Yuanyuan; Qin, Bin

2013-01-01

DNA microarray technology can measure the activities of tens of thousands of genes simultaneously, which provides an efficient way to diagnose cancer at the molecular level. Although this strategy has attracted significant research attention, most studies neglect an important problem, namely, that most DNA microarray datasets are skewed, which causes traditional learning algorithms to produce inaccurate results. Some studies have considered this problem, yet they merely focus on binary-class problem. In this paper, we dealt with multiclass imbalanced classification problem, as encountered in cancer DNA microarray, by using ensemble learning. We utilized one-against-all coding strategy to transform multiclass to multiple binary classes, each of them carrying out feature subspace, which is an evolving version of random subspace that generates multiple diverse training subsets. Next, we introduced one of two different correction technologies, namely, decision threshold adjustment or random undersampling, into each training subset to alleviate the damage of class imbalance. Specifically, support vector machine was used as base classifier, and a novel voting rule called counter voting was presented for making a final decision. Experimental results on eight skewed multiclass cancer microarray datasets indicate that unlike many traditional classification approaches, our methods are insensitive to class imbalance.

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

PubMed Central

Ramirez, Lisa S.; Wang, Jun

2016-01-01

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

PubMed Central

Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate

2012-01-01

Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (blaKPC) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 105 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship. PMID:23035190

Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples.

PubMed

Pine, P S; Boedigheimer, M; Rosenzweig, B A; Turpaz, Y; He, Y D; Delenstarr, G; Ganter, B; Jarnagin, K; Jones, W D; Reid, L H; Thompson, K L

2008-11-01

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

MGDB: crossing the marker genes of a user microarray with a database of public-microarrays marker genes.

PubMed

Huerta, Mario; Munyi, Marc; Expósito, David; Querol, Enric; Cedano, Juan

2014-06-15

The microarrays performed by scientific teams grow exponentially. These microarray data could be useful for researchers around the world, but unfortunately they are underused. To fully exploit these data, it is necessary (i) to extract these data from a repository of the high-throughput gene expression data like Gene Expression Omnibus (GEO) and (ii) to make the data from different microarrays comparable with tools easy to use for scientists. We have developed these two solutions in our server, implementing a database of microarray marker genes (Marker Genes Data Base). This database contains the marker genes of all GEO microarray datasets and it is updated monthly with the new microarrays from GEO. Thus, researchers can see whether the marker genes of their microarray are marker genes in other microarrays in the database, expanding the analysis of their microarray to the rest of the public microarrays. This solution helps not only to corroborate the conclusions regarding a researcher's microarray but also to identify the phenotype of different subsets of individuals under investigation, to frame the results with microarray experiments from other species, pathologies or tissues, to search for drugs that promote the transition between the studied phenotypes, to detect undesirable side effects of the treatment applied, etc. Thus, the researcher can quickly add relevant information to his/her studies from all of the previous analyses performed in other studies as long as they have been deposited in public repositories. Marker-gene database tool: http://ibb.uab.es/mgdb © The Author 2014. Published by Oxford University Press.

Thermodynamically optimal whole-genome tiling microarray design and validation.

PubMed

Cho, Hyejin; Chou, Hui-Hsien

2016-06-13

Microarray is an efficient apparatus to interrogate the whole transcriptome of species. Microarray can be designed according to annotated gene sets, but the resulted microarrays cannot be used to identify novel transcripts and this design method is not applicable to unannotated species. Alternatively, a whole-genome tiling microarray can be designed using only genomic sequences without gene annotations, and it can be used to detect novel RNA transcripts as well as known genes. The difficulty with tiling microarray design lies in the tradeoff between probe-specificity and coverage of the genome. Sequence comparison methods based on BLAST or similar software are commonly employed in microarray design, but they cannot precisely determine the subtle thermodynamic competition between probe targets and partially matched probe nontargets during hybridizations. Using the whole-genome thermodynamic analysis software PICKY to design tiling microarrays, we can achieve maximum whole-genome coverage allowable under the thermodynamic constraints of each target genome. The resulted tiling microarrays are thermodynamically optimal in the sense that all selected probes share the same melting temperature separation range between their targets and closest nontargets, and no additional probes can be added without violating the specificity of the microarray to the target genome. This new design method was used to create two whole-genome tiling microarrays for Escherichia coli MG1655 and Agrobacterium tumefaciens C58 and the experiment results validated the design.

Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Proudnikov, D.; Kirillov, E.; Chumakov, K.

2000-01-01

This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements.more » Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.« less

Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis

PubMed Central

2011-01-01

Background Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence in situ hybridization (FISH). Results Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23. Conclusions Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future. PMID:22087757

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

PubMed

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Identification of Escherichia coli O157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

PubMed Central

Swimley, Michelle S.; Taylor, Amber W.; Dawson, Erica D.

2011-01-01

Abstract Shiga toxin–producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100–1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains. PMID:21288130

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA

PubMed Central

Singh, Ruchira; Maganti, Rajanikanth J.; Jabba, Sairam V.; Wang, Martin; Deng, Glenn; Heath, Joe Don; Kurn, Nurith; Wangemann, Philine

2007-01-01

Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 μg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA. PMID:15613496

Undetected sex chromosome aneuploidy by chromosomal microarray.

PubMed

Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

2012-11-01

We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting. © 2012 John Wiley & Sons, Ltd.

PTM Microarray: Request for Year 3 Set-Aside Funds — EDRN Public Portal

Cancer.gov

We hypothesize that PTMs on proteins that are secreted by the breast will provide a more sensitive method for detecting breast cancer than analysis of the parent protein. We will antibody microarrays to have examine 9 circulating proteins, each of which is known to be actively secreted by the breast, for several structurally and functionally distinct PTMs. We will determine if these modified proteins have the potential to used in the early detection of breast cancer.

Derivation of Tissue-specific Functional Gene Sets to Aid Transcriptomic Analysis of Chemical Impacts on the Teleost Reproductive Axis.

EPA Science Inventory

Oligonucleotide microarrays are a powerful tool for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the poor power of microarray-based analyses to detect diffe...

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Microarray-integrated optoelectrofluidic immunoassay system

PubMed Central

Han, Dongsik

2016-01-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection. PMID:27190571

Microarray-integrated optoelectrofluidic immunoassay system.

PubMed

Han, Dongsik; Park, Je-Kyun

2016-05-01

A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets.

PubMed

Kim, Taehyung; Tyndel, Marc S; Huang, Haiming; Sidhu, Sachdev S; Bader, Gary D; Gfeller, David; Kim, Philip M

2012-03-01

Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

PubMed Central

Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

2014-01-01

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp. PMID:25469776

A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

PubMed

Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

2014-01-01

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification†

PubMed Central

Wu, Liyou; Liu, Xueduan; Schadt, Christopher W.; Zhou, Jizhong

2006-01-01

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment. PMID:16820490

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

PubMed

Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

2003-01-01

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

AccuTyping: new algorithms for automated analysis of data from high-throughput genotyping with oligonucleotide microarrays

PubMed Central

Hu, Guohong; Wang, Hui-Yun; Greenawalt, Danielle M.; Azaro, Marco A.; Luo, Minjie; Tereshchenko, Irina V.; Cui, Xiangfeng; Yang, Qifeng; Gao, Richeng; Shen, Li; Li, Honghua

2006-01-01

Microarray-based analysis of single nucleotide polymorphisms (SNPs) has many applications in large-scale genetic studies. To minimize the influence of experimental variation, microarray data usually need to be processed in different aspects including background subtraction, normalization and low-signal filtering before genotype determination. Although many algorithms are sophisticated for these purposes, biases are still present. In the present paper, new algorithms for SNP microarray data analysis and the software, AccuTyping, developed based on these algorithms are described. The algorithms take advantage of a large number of SNPs included in each assay, and the fact that the top and bottom 20% of SNPs can be safely treated as homozygous after sorting based on their ratios between the signal intensities. These SNPs are then used as controls for color channel normalization and background subtraction. Genotype calls are made based on the logarithms of signal intensity ratios using two cutoff values, which were determined after training the program with a dataset of ∼160 000 genotypes and validated by non-microarray methods. AccuTyping was used to determine >300 000 genotypes of DNA and sperm samples. The accuracy was shown to be >99%. AccuTyping can be downloaded from . PMID:16982644

Hidden Markov Model-Based CNV Detection Algorithms for Illumina Genotyping Microarrays.

PubMed

Seiser, Eric L; Innocenti, Federico

2014-01-01

Somatic alterations in DNA copy number have been well studied in numerous malignancies, yet the role of germline DNA copy number variation in cancer is still emerging. Genotyping microarrays generate allele-specific signal intensities to determine genotype, but may also be used to infer DNA copy number using additional computational approaches. Numerous tools have been developed to analyze Illumina genotype microarray data for copy number variant (CNV) discovery, although commonly utilized algorithms freely available to the public employ approaches based upon the use of hidden Markov models (HMMs). QuantiSNP, PennCNV, and GenoCN utilize HMMs with six copy number states but vary in how transition and emission probabilities are calculated. Performance of these CNV detection algorithms has been shown to be variable between both genotyping platforms and data sets, although HMM approaches generally outperform other current methods. Low sensitivity is prevalent with HMM-based algorithms, suggesting the need for continued improvement in CNV detection methodologies.

A Model-Based Joint Identification of Differentially Expressed Genes and Phenotype-Associated Genes

PubMed Central

Seo, Minseok; Shin, Su-kyung; Kwon, Eun-Young; Kim, Sung-Eun; Bae, Yun-Jung; Lee, Seungyeoun; Sung, Mi-Kyung; Choi, Myung-Sook; Park, Taesung

2016-01-01

Over the last decade, many analytical methods and tools have been developed for microarray data. The detection of differentially expressed genes (DEGs) among different treatment groups is often a primary purpose of microarray data analysis. In addition, association studies investigating the relationship between genes and a phenotype of interest such as survival time are also popular in microarray data analysis. Phenotype association analysis provides a list of phenotype-associated genes (PAGs). However, it is sometimes necessary to identify genes that are both DEGs and PAGs. We consider the joint identification of DEGs and PAGs in microarray data analyses. The first approach we used was a naïve approach that detects DEGs and PAGs separately and then identifies the genes in an intersection of the list of PAGs and DEGs. The second approach we considered was a hierarchical approach that detects DEGs first and then chooses PAGs from among the DEGs or vice versa. In this study, we propose a new model-based approach for the joint identification of DEGs and PAGs. Unlike the previous two-step approaches, the proposed method identifies genes simultaneously that are DEGs and PAGs. This method uses standard regression models but adopts different null hypothesis from ordinary regression models, which allows us to perform joint identification in one-step. The proposed model-based methods were evaluated using experimental data and simulation studies. The proposed methods were used to analyze a microarray experiment in which the main interest lies in detecting genes that are both DEGs and PAGs, where DEGs are identified between two diet groups and PAGs are associated with four phenotypes reflecting the expression of leptin, adiponectin, insulin-like growth factor 1, and insulin. Model-based approaches provided a larger number of genes, which are both DEGs and PAGs, than other methods. Simulation studies showed that they have more power than other methods. Through analysis of data from experimental microarrays and simulation studies, the proposed model-based approach was shown to provide a more powerful result than the naïve approach and the hierarchical approach. Since our approach is model-based, it is very flexible and can easily handle different types of covariates. PMID:26964035

ATMAD: robust image analysis for Automatic Tissue MicroArray De-arraying.

PubMed

Nguyen, Hoai Nam; Paveau, Vincent; Cauchois, Cyril; Kervrann, Charles

2018-04-19

Over the last two decades, an innovative technology called Tissue Microarray (TMA), which combines multi-tissue and DNA microarray concepts, has been widely used in the field of histology. It consists of a collection of several (up to 1000 or more) tissue samples that are assembled onto a single support - typically a glass slide - according to a design grid (array) layout, in order to allow multiplex analysis by treating numerous samples under identical and standardized conditions. However, during the TMA manufacturing process, the sample positions can be highly distorted from the design grid due to the imprecision when assembling tissue samples and the deformation of the embedding waxes. Consequently, these distortions may lead to severe errors of (histological) assay results when the sample identities are mismatched between the design and its manufactured output. The development of a robust method for de-arraying TMA, which localizes and matches TMA samples with their design grid, is therefore crucial to overcome the bottleneck of this prominent technology. In this paper, we propose an Automatic, fast and robust TMA De-arraying (ATMAD) approach dedicated to images acquired with brightfield and fluorescence microscopes (or scanners). First, tissue samples are localized in the large image by applying a locally adaptive thresholding on the isotropic wavelet transform of the input TMA image. To reduce false detections, a parametric shape model is considered for segmenting ellipse-shaped objects at each detected position. Segmented objects that do not meet the size and the roundness criteria are discarded from the list of tissue samples before being matched with the design grid. Sample matching is performed by estimating the TMA grid deformation under the thin-plate model. Finally, thanks to the estimated deformation, the true tissue samples that were preliminary rejected in the early image processing step are recognized by running a second segmentation step. We developed a novel de-arraying approach for TMA analysis. By combining wavelet-based detection, active contour segmentation, and thin-plate spline interpolation, our approach is able to handle TMA images with high dynamic, poor signal-to-noise ratio, complex background and non-linear deformation of TMA grid. In addition, the deformation estimation produces quantitative information to asset the manufacturing quality of TMAs.

Simplified Microarray Technique for Identifying mRNA in Rare Samples

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

Quality control of inkjet technology for DNA microarray fabrication.

PubMed

Pierik, Anke; Dijksman, Frits; Raaijmakers, Adrie; Wismans, Ton; Stapert, Henk

2008-12-01

A robust manufacturing process is essential to make high-quality DNA microarrays, especially for use in diagnostic tests. We investigated different failure modes of the inkjet printing process used to manufacture low-density microarrays. A single nozzle inkjet spotter was provided with two optical imaging systems, monitoring in real time the flight path of every droplet. If a droplet emission failure is detected, the printing process is automatically stopped. We analyzed over 1.3 million droplets. This information was used to investigate the performance of the inkjet system and to obtain detailed insight into the frequency and causes of jetting failures. Of all the substrates investigated, 96.2% were produced without any system or jetting failures. In 1.6% of the substrates, droplet emission failed and was correctly identified. Appropriate measures could then be taken to get the process back on track. In 2.2%, the imaging systems failed while droplet emission occurred correctly. In 0.1% of the substrates, droplet emission failure that was not timely detected occurred. Thus, the overall yield of the microarray manufacturing process was 99.9%, which is highly acceptable for prototyping.

The Choice of the Filtering Method in Microarrays Affects the Inference Regarding Dosage Compensation of the Active X-Chromosome

PubMed Central

Zeller, Tanja; Wild, Philipp S.; Truong, Vinh; Trégouët, David-Alexandre; Munzel, Thomas; Ziegler, Andreas; Cambien, François; Blankenberg, Stefan; Tiret, Laurence

2011-01-01

Background The hypothesis of dosage compensation of genes of the X chromosome, supported by previous microarray studies, was recently challenged by RNA-sequencing data. It was suggested that microarray studies were biased toward an over-estimation of X-linked expression levels as a consequence of the filtering of genes below the detection threshold of microarrays. Methodology/Principal Findings To investigate this hypothesis, we used microarray expression data from circulating monocytes in 1,467 individuals. In total, 25,349 and 1,156 probes were unambiguously assigned to autosomes and the X chromosome, respectively. Globally, there was a clear shift of X-linked expressions toward lower levels than autosomes. We compared the ratio of expression levels of X-linked to autosomal transcripts (X∶AA) using two different filtering methods: 1. gene expressions were filtered out using a detection threshold irrespective of gene chromosomal location (the standard method in microarrays); 2. equal proportions of genes were filtered out separately on the X and on autosomes. For a wide range of filtering proportions, the X∶AA ratio estimated with the first method was not significantly different from 1, the value expected if dosage compensation was achieved, whereas it was significantly lower than 1 with the second method, leading to the rejection of the hypothesis of dosage compensation. We further showed in simulated data that the choice of the most appropriate method was dependent on biological assumptions regarding the proportion of actively expressed genes on the X chromosome comparative to the autosomes and the extent of dosage compensation. Conclusion/Significance This study shows that the method used for filtering out lowly expressed genes in microarrays may have a major impact according to the hypothesis investigated. The hypothesis of dosage compensation of X-linked genes cannot be firmly accepted or rejected using microarray-based data. PMID:21912656

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

PubMed

Gehring, Andrew G; Brewster, Jeffrey D; He, Yiping; Irwin, Peter L; Paoli, George C; Simons, Tawana; Tu, Shu-I; Uknalis, Joseph

2015-12-04

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples.

PubMed

Sanchis, Ana; Salvador, J-Pablo; Campbell, Katrina; Elliott, Christopher T; Shelver, Weilin L; Li, Qing X; Marco, M-Pilar

2018-07-01

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17β-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19 ± 0,06 µg L -1 ; sulfapyridine, 0.17 ± 0.07 µg L -1 ; chloramphenicol, 0.11 ± 0.03 µg L -1 ; BDE-47, 2.71 ± 1.13 µg L -1 ; 17β-estradiol, 0.94 ± 0.30 µg L -1 and domoic acid, 1.71 ± 0.30 µg L -1 ). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was < 20% in all the cases. No sample pre-treatment was necessary, and the results could be obtained in just 1 h 30 min. The microarray shows high sample throughput capabilities, being able to measure simultaneously more than 68 samples and screen them for a significant number of chemical contaminants of interest in environmental screening programs. Copyright © 2018 Elsevier B.V. All rights reserved.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways. PMID:16608515

Spotting effect in microarray experiments

PubMed Central

Mary-Huard, Tristan; Daudin, Jean-Jacques; Robin, Stéphane; Bitton, Frédérique; Cabannes, Eric; Hilson, Pierre

2004-01-01

Background Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array. Results Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure. Conclusions The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis. PMID:15151695

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

PubMed

Wang, Zheng; Vora, Gary J; Stenger, David A

2004-07-01

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

Highly sensitive detection of multiple tumor markers for lung cancer using gold nanoparticle probes and microarrays.

PubMed

Gao, Wanlei; Wang, Wentao; Yao, Shihua; Wu, Shan; Zhang, Honglian; Zhang, Jishen; Jing, Fengxiang; Mao, Hongju; Jin, Qinghui; Cong, Hui; Jia, Chunping; Zhang, Guojun; Zhao, Jianlong

2017-03-15

Assay of multiple serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE), is important for the early diagnosis of lung cancer. Dickkopf-1 (DKK1), a novel serological and histochemical biomarker, was recently reported to be preferentially expressed in lung cancer. Four target proteins were sandwiched by capture antibodies attached to microarrays and detection antibodies carried on modified gold nanoparticles. Optical signals generated by the sandwich structures were amplified by gold deposition with HAuCl 4 and H 2 O 2 , and were observable by microscopy or the naked eye. The four tumor markers were subsequently measured in 106 lung cancer patients and 42 healthy persons. The assay was capable of detecting multiple biomarkers in serum sample at concentration of <1 ng mL -1 in 1 h. Combined detection of the four tumor markers highly improved the sensitivity (to 87.74%) for diagnosis of lung cancer compared with sensitivity of single markers. A rapid, highly sensitive co-detection method for multiple biomarkers based on gold nanoparticles and microarrays was developed. In clinical use, it would be expected to improve the early diagnosis of lung cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

PubMed Central

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathy L.; Marks, James D.; Varnum, Susan M.

2012-01-01

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples. PMID:22935296

Evaluation of the efficacy of constitutional array-based comparative genomic hybridization in the diagnosis of aneuploidy using genomic and amplified DNA.

PubMed

Tan, Niap H; Palmer, Rodger; Wang, Rubin

2010-02-01

Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance. The present study investigated the efficacy of constitutional microarray in the diagnosis of trisomy. Test samples included genomic DNA from trisomic cell lines, amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. DNA amplification was achieved by means of multiple displacement amplification (MDA) over 16 h. The trisomic and sex chromosomes copy number imbalances in the genomic DNA were correctly identified by the constitutional microarrays. However, there was a failure to detect the trisomy in the amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. Using carefully selected clones, Spectral Genomics constitutional microarray was able to detect the chromosomal copy number imbalances in genomic DNA without the confounding effects of CNV. The diagnostic failure in amplified DNA samples could be attributed to the amplification process. The MDA duration of 16 h generated excessive amount of biases and shortening the duration might minimize the problem.

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

PubMed Central

Le Berre, Véronique; Trévisiol, Emmanuelle; Dagkessamanskaia, Adilia; Sokol, Serguei; Caminade, Anne-Marie; Majoral, Jean Pierre; Meunier, Bernard; François, Jean

2003-01-01

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ∼100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations. PMID:12907740

RNA Expression Profiles from Blood for the Diagnosis of Stroke and its Causes

PubMed Central

Sharp, Frank R; Jickling, Glen C; Stamova, Boryana; Tian, Yingfang; Zhan, Xinhua; Ander, Bradley P; Cox, Christopher; Kuczynski, Beth; Liu, DaZhi

2013-01-01

A blood test to detect stroke and its causes would be particularly useful in babies, young children, and patients in intensive care units, and for emergencies when imaging is difficult to obtain or unavailable. Using whole genome microarrays, we first showed specific gene expression profiles in rats 24 hours after ischemic and hemorrhagic stroke, hypoxia, and hypoglycemia. These proof-of-principle studies revealed that groups of genes (called gene profiles) can distinguish ischemic stroke patients from controls 3 hours to 24 hours after the strokes. In addition, gene expression profiles have been developed that distinguish stroke due to large-vessel atherosclerosis from cardioembolic stroke. These profiles will be useful for predicting the causes of cryptogenic stroke. Our results in adults suggest similar diagnostic tools could be developed for children. PMID:21636778

A hybrid approach to device integration on a genetic analysis platform

NASA Astrophysics Data System (ADS)

Brennan, Des; Jary, Dorothee; Kurg, Ants; Berik, Evgeny; Justice, John; Aherne, Margaret; Macek, Milan; Galvin, Paul

2012-10-01

Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization.

Experimental design for three-color and four-color gene expression microarrays.

PubMed

Woo, Yong; Krueger, Winfried; Kaur, Anupinder; Churchill, Gary

2005-06-01

Three-color microarrays, compared with two-color microarrays, can increase design efficiency and power to detect differential expression without additional samples and arrays. Furthermore, three-color microarray technology is currently available at a reasonable cost. Despite the potential advantages, clear guidelines for designing and analyzing three-color experiments do not exist. We propose a three- and a four-color cyclic design (loop) and a complementary graphical representation to help design experiments that are balanced, efficient and robust to hybridization failures. In theory, three-color loop designs are more efficient than two-color loop designs. Experiments using both two- and three-color platforms were performed in parallel and their outputs were analyzed using linear mixed model analysis in R/MAANOVA. These results demonstrate that three-color experiments using the same number of samples (and fewer arrays) will perform as efficiently as two-color experiments. The improved efficiency of the design is somewhat offset by a reduced dynamic range and increased variability in the three-color experimental system. This result suggests that, with minor technological improvements, three-color microarrays using loop designs could detect differential expression more efficiently than two-color loop designs. http://www.jax.org/staff/churchill/labsite/software Multicolor cyclic design construction methods and examples along with additional results of the experiment are provided at http://www.jax.org/staff/churchill/labsite/pubs/yong.

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

PubMed

Hartmann, Luise; Stephenson, Christine F; Verkamp, Stephanie R; Johnson, Krystal R; Burnworth, Bettina; Hammock, Kelle; Brodersen, Lisa Eidenschink; de Baca, Monica E; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2014-12-01

Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry.

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

PubMed Central

Pinzani, Pamela; Mancini, Irene; Vinci, Serena; Chiari, Marcella; Orlando, Claudio; Cremonesi, Laura; Ferrari, Maurizio

2013-01-01

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy. PMID:23536897

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides.

PubMed

De Marni, Marzia L; Monegal, Ana; Venturini, Samuele; Vinati, Simone; Carbone, Roberta; de Marco, Ario

2012-02-01

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs. Copyright © 2011. Published by Elsevier Inc.

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

PubMed

Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

2018-03-12

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Prenatal chromosomal microarray analysis in fetuses with congenital heart disease: a prospective cohort study.

PubMed

Wang, Yan; Cao, Li; Liang, Dong; Meng, Lulu; Wu, Yun; Qiao, Fengchang; Ji, Xiuqing; Luo, Chunyu; Zhang, Jingjing; Xu, Tianhui; Yu, Bin; Wang, Leilei; Wang, Ting; Pan, Qiong; Ma, Dingyuan; Hu, Ping; Xu, Zhengfeng

2018-02-01

Currently, chromosomal microarray analysis is considered the first-tier test in pediatric care and prenatal diagnosis. However, the diagnostic yield of chromosomal microarray analysis for prenatal diagnosis of congenital heart disease has not been evaluated based on a large cohort. Our aim was to evaluate the clinical utility of chromosomal microarray as the first-tier test for chromosomal abnormalities in fetuses with congenital heart disease. In this prospective study, 602 prenatal cases of congenital heart disease were investigated using single nucleotide polymorphism array over a 5-year period. Overall, pathogenic chromosomal abnormalities were identified in 125 (20.8%) of 602 prenatal cases of congenital heart disease, with 52.0% of them being numerical chromosomal abnormalities. The detection rates of likely pathogenic copy number variations and variants of uncertain significance were 1.3% and 6.0%, respectively. The detection rate of pathogenic chromosomal abnormalities in congenital heart disease plus additional structural anomalies (48.9% vs 14.3%, P < .0001) or intrauterine growth retardation group (50.0% vs 14.3%, P = .044) was significantly higher than that in isolated congenital heart disease group. Additionally, the detection rate in congenital heart disease with additional structural anomalies group was significantly higher than that in congenital heart disease with soft markers group (48.9% vs 19.8%, P < .0001). No significant difference was observed in the detection rates between congenital heart disease with additional structural anomalies and congenital heart disease with intrauterine growth retardation groups (48.9% vs 50.0%), congenital heart disease with soft markers and congenital heart disease with intrauterine growth retardation groups (19.8% vs 50.0%), or congenital heart disease with soft markers and isolated congenital heart disease groups (19.8% vs 14.3%). The detection rate in fetuses with congenital heart disease plus mild ventriculomegaly was significantly higher than in those with other types of soft markers (50.0% vs 15.6%, P < .05). Our study suggests chromosomal microarray analysis is a reliable and high-resolution technology and should be used as the first-tier test for prenatal diagnosis of congenital heart disease in clinical practice. Copyright © 2017 Elsevier Inc. All rights reserved.

Experimental analysis of oligonucleotide microarray design criteria to detect deletions by comparative genomic hybridization.

PubMed

Flibotte, Stephane; Moerman, Donald G

2008-10-21

Microarray comparative genomic hybridization (CGH) is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans) the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10 degrees C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy. We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species.

A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

PubMed Central

Gehring, Andrew; He, Xiaohua; Fratamico, Pina; Lee, Joseph; Bagi, Lori; Brewster, Jeffrey; Paoli, George; He, Yiping; Xie, Yanping; Skinner, Craig; Barnett, Charlie; Harris, Douglas

2014-01-01

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef. PMID:24921195

A dynamic bead-based microarray for parallel DNA detection

NASA Astrophysics Data System (ADS)

Sochol, R. D.; Casavant, B. P.; Dueck, M. E.; Lee, L. P.; Lin, L.

2011-05-01

A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening.

Electronic hybridization detection in microarray format and DNA genotyping

NASA Astrophysics Data System (ADS)

Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

2014-02-01

We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

Electronic hybridization detection in microarray format and DNA genotyping

PubMed Central

Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

2014-01-01

We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device. PMID:24569823

Microarray Detection of Duplex and Triplex DNA Binders with DNA-Modified Gold Nanoparticles

PubMed Central

Lytton-Jean, Abigail K. R.; Han, Min Su; Mirkin, Chad A.

2008-01-01

We have designed a chip-based assay, using microarray technology, for determining the relative binding affinities of duplex and triplex DNA binders. This assay combines the high discrimination capabilities afforded by DNA-modified Au nanoparticles with the high-throughput capabilities of DNA microarrays. The detection and screening of duplex DNA binders are important because these molecules, in many cases, are potential anticancer agents as well as toxins. Triplex DNA binders are also promising drug candidates. These molecules, in conjunction with triplex forming oligonucleotides, could potentially be used to achieve control of gene expression by interfering with transcription factors that bind to DNA. Therefore, the ability to screen for these molecules in a high-throughput fashion could dramatically improve the drug screening process. The assay reported here provides excellent discrimination between strong, intermediate, and weak duplex and triplex DNA binders in a high-throughput fashion. PMID:17614366

Shrink-induced silica multiscale structures for enhanced fluorescence from DNA microarrays.

PubMed

Sharma, Himanshu; Wood, Jennifer B; Lin, Sophia; Corn, Robert M; Khine, Michelle

2014-09-23

We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30-45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays.

Shrink-Induced Silica Multiscale Structures for Enhanced Fluorescence from DNA Microarrays

PubMed Central

2015-01-01

We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30–45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays. PMID:25191785

MULTI-K: accurate classification of microarray subtypes using ensemble k-means clustering

PubMed Central

Kim, Eun-Youn; Kim, Seon-Young; Ashlock, Daniel; Nam, Dougu

2009-01-01

Background Uncovering subtypes of disease from microarray samples has important clinical implications such as survival time and sensitivity of individual patients to specific therapies. Unsupervised clustering methods have been used to classify this type of data. However, most existing methods focus on clusters with compact shapes and do not reflect the geometric complexity of the high dimensional microarray clusters, which limits their performance. Results We present a cluster-number-based ensemble clustering algorithm, called MULTI-K, for microarray sample classification, which demonstrates remarkable accuracy. The method amalgamates multiple k-means runs by varying the number of clusters and identifies clusters that manifest the most robust co-memberships of elements. In addition to the original algorithm, we newly devised the entropy-plot to control the separation of singletons or small clusters. MULTI-K, unlike the simple k-means or other widely used methods, was able to capture clusters with complex and high-dimensional structures accurately. MULTI-K outperformed other methods including a recently developed ensemble clustering algorithm in tests with five simulated and eight real gene-expression data sets. Conclusion The geometric complexity of clusters should be taken into account for accurate classification of microarray data, and ensemble clustering applied to the number of clusters tackles the problem very well. The C++ code and the data sets tested are available from the authors. PMID:19698124

Distributional fold change test – a statistical approach for detecting differential expression in microarray experiments

PubMed Central

2012-01-01

Background Because of the large volume of data and the intrinsic variation of data intensity observed in microarray experiments, different statistical methods have been used to systematically extract biological information and to quantify the associated uncertainty. The simplest method to identify differentially expressed genes is to evaluate the ratio of average intensities in two different conditions and consider all genes that differ by more than an arbitrary cut-off value to be differentially expressed. This filtering approach is not a statistical test and there is no associated value that can indicate the level of confidence in the designation of genes as differentially expressed or not differentially expressed. At the same time the fold change by itself provide valuable information and it is important to find unambiguous ways of using this information in expression data treatment. Results A new method of finding differentially expressed genes, called distributional fold change (DFC) test is introduced. The method is based on an analysis of the intensity distribution of all microarray probe sets mapped to a three dimensional feature space composed of average expression level, average difference of gene expression and total variance. The proposed method allows one to rank each feature based on the signal-to-noise ratio and to ascertain for each feature the confidence level and power for being differentially expressed. The performance of the new method was evaluated using the total and partial area under receiver operating curves and tested on 11 data sets from Gene Omnibus Database with independently verified differentially expressed genes and compared with the t-test and shrinkage t-test. Overall the DFC test performed the best – on average it had higher sensitivity and partial AUC and its elevation was most prominent in the low range of differentially expressed features, typical for formalin-fixed paraffin-embedded sample sets. Conclusions The distributional fold change test is an effective method for finding and ranking differentially expressed probesets on microarrays. The application of this test is advantageous to data sets using formalin-fixed paraffin-embedded samples or other systems where degradation effects diminish the applicability of correlation adjusted methods to the whole feature set. PMID:23122055

Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

PubMed Central

Loy, Alexander; Lehner, Angelika; Lee, Natuschka; Adamczyk, Justyna; Meier, Harald; Ernst, Jens; Schleifer, Karl-Heinz; Wagner, Michael

2002-01-01

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB). PMID:12324358

Development of oligonucleotide microarrays for simultaneous multi-species identification of Phellinus tree-pathogenic fungi.

PubMed

Tzean, Yuh; Shu, Po-Yao; Liou, Ruey-Fen; Tzean, Shean-Shong

2016-03-01

Polyporoid Phellinus fungi are ubiquitously present in the environment and play an important role in shaping forest ecology. Several species of Phellinus are notorious pathogens that can affect a broad variety of tree species in forest, plantation, orchard and urban habitats; however, current detection methods are overly complex and lack the sensitivity required to identify these pathogens at the species level in a timely fashion for effective infestation control. Here, we describe eight oligonucleotide microarray platforms for the simultaneous and specific detection of 17 important Phellinus species, using probes generated from the internal transcribed spacer regions unique to each species. The sensitivity, robustness and efficiency of this Phellinus microarray system was subsequently confirmed against template DNA from two key Phellinus species, as well as field samples collected from tree roots, trunks and surrounding soil. This system can provide early, specific and convenient detection of Phellinus species for forestry, arboriculture and quarantine inspection, and could potentially help to mitigate the environmental and economic impact of Phellinus-related diseases. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Prenatal diagnosis of fetuses with increased nuchal translucency using an approach based on quantitative fluorescent polymerase chain reaction and genomic microarray.

PubMed

Pan, Min; Han, Jin; Zhen, Li; Yang, Xin; Li, Ru; Liao, Can; Li, Dong-Zhi

2016-02-01

To assess the clinical value of prenatal diagnosis of fetuses with increased nuchal translucency (NT) using an approach based on quantitative fluorescent polymerase chain reaction (QF-PCR) and chromosomal microarray (CMA). From January 2013 to October 2014, we included 175 pregnancies with fetal NT ≥ 3.5mm at 11-13 weeks' gestation who received chorionic villus sampling. QF-PCR was first used to rapidly detect common aneuploidies. The cases with a normal QF-PCR result were analyzed by CMA. Of the 175 cases, common aneuploidies were detected by QF-PCR in 53 (30.2%) cases (30 cases of trisomy 21, 12 cases of monosomy X, 7 cases of trisomy 18, 3 cases of trisomy 13 and 1 case of 47, XXY). Among the 122 cases with a normal QF-PCR result, microarray detected additional pathogenic copy number variants (CNVs) in 5.7% (7/122) of cases. Four cases would have expected to be detectable by conventional karyotyping because of large deletions/duplications (>10 Mb), leaving three cases (2.5%; 3/118) with pathogenic CNVs only detectable by CMA. It is rational to use a diagnostic strategy in which CMA is preceded by the less expensive, rapid, QF-PCR to detect common aneuploidies. CMA allows detection of a number of pathogenic chromosomal aberrations in fetuses with a high NT. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Microarray-Based Mapping for the Detection of Molecular Markers in Response to Aspergillus flavus Infection in Susceptible and Resistant Maize Lines

USDA-ARS?s Scientific Manuscript database

The objectives of this study were (1) to evaluate differential gene expression levels for resistance to A. flavus kernel infection in susceptible (Va35) and resistant (Mp313E) maize lines using Oligonucleotide and cDNA microarray analysis, (2) to evaluate differences in A. flavus accumulation betwee...

A Quick and Parallel Analytical Method Based on Quantum Dots Labeling for ToRCH-Related Antibodies

NASA Astrophysics Data System (ADS)

Yang, Hao; Guo, Qing; He, Rong; Li, Ding; Zhang, Xueqing; Bao, Chenchen; Hu, Hengyao; Cui, Daxiang

2009-12-01

Quantum dot is a special kind of nanomaterial composed of periodic groups of II-VI, III-V or IV-VI materials. Their high quantum yield, broad absorption with narrow photoluminescence spectra and high resistance to photobleaching, make them become a promising labeling substance in biological analysis. Here, we report a quick and parallel analytical method based on quantum dots for ToRCH-related antibodies including Toxoplasma gondii, Rubella virus, Cytomegalovirus and Herpes simplex virus type 1 (HSV1) and 2 (HSV2). Firstly, we fabricated the microarrays with the five kinds of ToRCH-related antigens and used CdTe quantum dots to label secondary antibody and then analyzed 100 specimens of randomly selected clinical sera from obstetric outpatients. The currently prevalent enzyme-linked immunosorbent assay (ELISA) kits were considered as “golden standard” for comparison. The results show that the quantum dots labeling-based ToRCH microarrays have comparable sensitivity and specificity with ELISA. Besides, the microarrays hold distinct advantages over ELISA test format in detection time, cost, operation and signal stability. Validated by the clinical assay, our quantum dots-based ToRCH microarrays have great potential in the detection of ToRCH-related pathogens.

High density DNA microarrays: algorithms and biomedical applications.

PubMed

Liu, Wei-Min

2004-08-01

DNA microarrays are devices capable of detecting the identity and abundance of numerous DNA or RNA segments in samples. They are used for analyzing gene expressions, identifying genetic markers and detecting mutations on a genomic scale. The fundamental chemical mechanism of DNA microarrays is the hybridization between probes and targets due to the hydrogen bonds of nucleotide base pairing. Since the cross hybridization is inevitable, and probes or targets may form undesirable secondary or tertiary structures, the microarray data contain noise and depend on experimental conditions. It is crucial to apply proper statistical algorithms to obtain useful signals from noisy data. After we obtained the signals of a large amount of probes, we need to derive the biomedical information such as the existence of a transcript in a cell, the difference of expression levels of a gene in multiple samples, and the type of a genetic marker. Furthermore, after the expression levels of thousands of genes or the genotypes of thousands of single nucleotide polymorphisms are determined, it is usually important to find a small number of genes or markers that are related to a disease, individual reactions to drugs, or other phenotypes. All these applications need careful data analyses and reliable algorithms.

Development and validation of a microarray for the investigation of the CAZymes encoded by the human gut microbiome.

PubMed

El Kaoutari, Abdessamad; Armougom, Fabrice; Leroy, Quentin; Vialettes, Bernard; Million, Matthieu; Raoult, Didier; Henrissat, Bernard

2013-01-01

Distal gut bacteria play a pivotal role in the digestion of dietary polysaccharides by producing a large number of carbohydrate-active enzymes (CAZymes) that the host otherwise does not produce. We report here the design of a custom microarray that we used to spot non-redundant DNA probes for more than 6,500 genes encoding glycoside hydrolases and lyases selected from 174 reference genomes from distal gut bacteria. The custom microarray was tested and validated by the hybridization of bacterial DNA extracted from the stool samples of lean, obese and anorexic individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the finding that a gene encoding a GH6-family cellulase was present in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding CAZome core containing 46 families of glycoside hydrolases and polysaccharide lyases, which suggests the functional stability of the gut microbiota despite large taxonomical variations between individuals.

Portable, stand-off spectral imaging camera for detection of effluents and residues

NASA Astrophysics Data System (ADS)

Goldstein, Neil; St. Peter, Benjamin; Grot, Jonathan; Kogan, Michael; Fox, Marsha; Vujkovic-Cvijin, Pajo; Penny, Ryan; Cline, Jason

2015-06-01

A new, compact and portable spectral imaging camera, employing a MEMs-based encoded imaging approach, has been built and demonstrated for detection of hazardous contaminants including gaseous effluents and solid-liquid residues on surfaces. The camera is called the Thermal infrared Reconfigurable Analysis Camera for Effluents and Residues (TRACER). TRACER operates in the long wave infrared and has the potential to detect a wide variety of materials with characteristic spectral signatures in that region. The 30 lb. camera is tripod mounted and battery powered. A touch screen control panel provides a simple user interface for most operations. The MEMS spatial light modulator is a Texas Instruments Digital Microarray Array with custom electronics and firmware control. Simultaneous 1D-spatial and 1Dspectral dimensions are collected, with the second spatial dimension obtained by scanning the internal spectrometer slit. The sensor can be configured to collect data in several modes including full hyperspectral imagery using Hadamard multiplexing, panchromatic thermal imagery, and chemical-specific contrast imagery, switched with simple user commands. Matched filters and other analog filters can be generated internally on-the-fly and applied in hardware, substantially reducing detection time and improving SNR over HSI software processing, while reducing storage requirements. Results of preliminary instrument evaluation and measurements of flame exhaust are presented.

Systematic spatial bias in DNA microarray hybridization is caused by probe spot position-dependent variability in lateral diffusion.

PubMed

Steger, Doris; Berry, David; Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

2011-01-01

The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization.

Systematic Spatial Bias in DNA Microarray Hybridization Is Caused by Probe Spot Position-Dependent Variability in Lateral Diffusion

PubMed Central

Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

2011-01-01

Background The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. Methodology/Principal Findings This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Conclusions Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. PMID:21858215

[Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

PubMed

Wang, Hong; Bi, Yongyi; Tao, Ning; Wang, Chunhong

2005-08-01

To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

Array for detecting microbes

DOEpatents

Andersen, Gary L.; DeSantis, Todd D.

2014-07-08

The present embodiments relate to an array system for detecting and identifying biomolecules and organisms. More specifically, the present embodiments relate to an array system comprising a microarray configured to simultaneously detect a plurality of organisms in a sample at a high confidence level.

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

PubMed

McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

2015-07-01

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Is this the real time for genomics?

PubMed

Guarnaccia, Maria; Gentile, Giulia; Alessi, Enrico; Schneider, Claudio; Petralia, Salvatore; Cavallaro, Sebastiano

2014-01-01

In the last decades, molecular biology has moved from gene-by-gene analysis to more complex studies using a genome-wide scale. Thanks to high-throughput genomic technologies, such as microarrays and next-generation sequencing, a huge amount of information has been generated, expanding our knowledge on the genetic basis of various diseases. Although some of this information could be transferred to clinical diagnostics, the technologies available are not suitable for this purpose. In this review, we will discuss the drawbacks associated with the use of traditional DNA microarrays in diagnostics, pointing out emerging platforms that could overcome these obstacles and offer a more reproducible, qualitative and quantitative multigenic analysis. New miniaturized and automated devices, called Lab-on-Chip, begin to integrate PCR and microarray on the same platform, offering integrated sample-to-result systems. The introduction of this kind of innovative devices may facilitate the transition of genome-based tests into clinical routine. Copyright © 2014. Published by Elsevier Inc.

PhylArray: phylogenetic probe design algorithm for microarray.

PubMed

Militon, Cécile; Rimour, Sébastien; Missaoui, Mohieddine; Biderre, Corinne; Barra, Vincent; Hill, David; Moné, Anne; Gagne, Geneviève; Meier, Harald; Peyretaillade, Eric; Peyret, Pierre

2007-10-01

Microbial diversity is still largely unknown in most environments, such as soils. In order to get access to this microbial 'black-box', the development of powerful tools such as microarrays are necessary. However, the reliability of this approach relies on probe efficiency, in particular sensitivity, specificity and explorative power, in order to obtain an image of the microbial communities that is close to reality. We propose a new probe design algorithm that is able to select microarray probes targeting SSU rRNA at any phylogenetic level. This original approach, implemented in a program called 'PhylArray', designs a combination of degenerate and non-degenerate probes for each target taxon. Comparative experimental evaluations indicate that probes designed with PhylArray yield a higher sensitivity and specificity than those designed by conventional approaches. Applying the combined PhyArray/GoArrays strategy helps to optimize the hybridization performance of short probes. Finally, hybridizations with environmental targets have shown that the use of the PhylArray strategy can draw attention to even previously unknown bacteria.

16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis.

PubMed

Topcuoglu, Nursen; Kulekci, Guven

2015-10-01

DNA microarray analysis is a computer based technology, that a reverse capture, which targets 10 periodontal bacteria (ParoCheck) is available for rapid semi-quantitative determination. The aim of this three-year retrospective study was to display the microarray analysis results for the subgingival biofilm samples taken from patient cases diagnosed with different forms of periodontitis. A total of 84 patients with generalized aggressive periodontitis (GAP,n:29), generalized chronic periodontitis (GCP, n:25), peri-implantitis (PI,n:14), localized aggressive periodontitis (LAP,n:8) and refractory chronic periodontitis (RP,n:8) were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory. The subgingival biofilm samples were analyzed by the microarray-based identification of 10 selected species. All the tested species were detected in the samples. The red complex bacteria were the most prevalent with very high levels in all groups. Fusobacterium nucleatum was detected in all samples at high levels. The green and blue complex bacteria were less prevalent compared with red and orange complex, except Aggregatibacter actinomycetemcomitas was detected in all LAP group. Positive correlations were found within all the red complex bacteria and between red and orange complex bacteria especially in GCP and GAP groups. Parocheck enables to monitoring of periodontal pathogens in all forms of periodontal disease and can be alternative to other guiding and reliable microbiologic tests. Copyright © 2015 Elsevier Ltd. All rights reserved.

Application of chemical arrays in screening elastase inhibitors.

PubMed

Gao, Feng; Du, Guan-Hua

2006-06-01

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).

Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens.

PubMed

Liebe, Sebastian; Christ, Daniela S; Ehricht, Ralf; Varrelmann, Mark

2016-01-01

Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present.

A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray

PubMed Central

Sachse, Konrad; Ziegler, Ute; Keller, Markus

2017-01-01

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented. PMID:28626758

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.

2012-11-15

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL)more » for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.« less

A segmentation/clustering model for the analysis of array CGH data.

PubMed

Picard, F; Robin, S; Lebarbier, E; Daudin, J-J

2007-09-01

Microarray-CGH (comparative genomic hybridization) experiments are used to detect and map chromosomal imbalances. A CGH profile can be viewed as a succession of segments that represent homogeneous regions in the genome whose representative sequences share the same relative copy number on average. Segmentation methods constitute a natural framework for the analysis, but they do not provide a biological status for the detected segments. We propose a new model for this segmentation/clustering problem, combining a segmentation model with a mixture model. We present a new hybrid algorithm called dynamic programming-expectation maximization (DP-EM) to estimate the parameters of the model by maximum likelihood. This algorithm combines DP and the EM algorithm. We also propose a model selection heuristic to select the number of clusters and the number of segments. An example of our procedure is presented, based on publicly available data sets. We compare our method to segmentation methods and to hidden Markov models, and we show that the new segmentation/clustering model is a promising alternative that can be applied in the more general context of signal processing.

Immunosignature: Serum Antibody Profiling for Cancer Diagnostics.

PubMed

Chapoval, Andrei I; Legutki, J Bart; Stafford, Philip; Trebukhov, Andrey V; Johnston, Stephen A; Shoikhet, Yakov N; Lazarev, Alexander F

2015-01-01

Biomarkers for preclinical diagnosis of cancer are valuable tools for detection of malignant tumors at early stages in groups at risk and screening healthy people, as well as monitoring disease recurrence after treatment of cancer. However the complexity of the body's response to the pathological processes makes it virtually impossible to evaluate this response to the development of the disease using a single biomarker that is present in the serum at low concentrations. An alternative approach to standard biomarker analysis is called immunosignature. Instead of going after biomarkers themselves this approach rely on the analysis of the humoral immune response to molecular changes associated with the development of pathological processes. It is known that antibodies are produced in response to proteins expressed during cancer development. Accordingly, the changes in antibody repertoire associated with tumor growth can serve as biomarkers of cancer. Immunosignature is a highly sensitive method for antibody repertoire analysis utilizing high density peptide microarrays. In the present review we discuss modern methods for antibody detection, as well as describe the principles and applications of immunosignature in research and clinical practice.

An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.

PubMed

Schüler, Susann; Wenz, Ingrid; Wiederanders, B; Slickers, P; Ehricht, R

2006-06-12

Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures. In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well. As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.

Profiling In Situ Microbial Community Structure with an Amplification Microarray

PubMed Central

Knickerbocker, Christopher; Bryant, Lexi; Golova, Julia; Wiles, Cory; Williams, Kenneth H.; Peacock, Aaron D.; Long, Philip E.

2013-01-01

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO3−) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO3, but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications. PMID:23160129

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

PubMed

Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

PubMed Central

Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide. PMID:20210464

A biomimetic algorithm for the improved detection of microarray features

NASA Astrophysics Data System (ADS)

Nicolau, Dan V., Jr.; Nicolau, Dan V.; Maini, Philip K.

2007-02-01

One the major difficulties of microarray technology relate to the processing of large and - importantly - error-loaded images of the dots on the chip surface. Whatever the source of these errors, those obtained in the first stage of data acquisition - segmentation - are passed down to the subsequent processes, with deleterious results. As it has been demonstrated recently that biological systems have evolved algorithms that are mathematically efficient, this contribution attempts to test an algorithm that mimics a bacterial-"patented" algorithm for the search of available space and nutrients to find, "zero-in" and eventually delimitate the features existent on the microarray surface.

Generalized Correlation Coefficient for Non-Parametric Analysis of Microarray Time-Course Data.

PubMed

Tan, Qihua; Thomassen, Mads; Burton, Mark; Mose, Kristian Fredløv; Andersen, Klaus Ejner; Hjelmborg, Jacob; Kruse, Torben

2017-06-06

Modeling complex time-course patterns is a challenging issue in microarray study due to complex gene expression patterns in response to the time-course experiment. We introduce the generalized correlation coefficient and propose a combinatory approach for detecting, testing and clustering the heterogeneous time-course gene expression patterns. Application of the method identified nonlinear time-course patterns in high agreement with parametric analysis. We conclude that the non-parametric nature in the generalized correlation analysis could be an useful and efficient tool for analyzing microarray time-course data and for exploring the complex relationships in the omics data for studying their association with disease and health.

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia.

PubMed

Schönmann, Susan; Loy, Alexander; Wimmersberger, Céline; Sobek, Jens; Aquino, Catharine; Vandamme, Peter; Frey, Beat; Rehrauer, Hubert; Eberl, Leo

2009-04-01

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia, an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia, Pandoraea, Ralstonia and Limnobacter. Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool Phylo- Detect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmiumcontaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.

Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli.

PubMed

Geue, Lutz; Stieber, Bettina; Monecke, Stefan; Engelmann, Ines; Gunzer, Florian; Slickers, Peter; Braun, Sascha D; Ehricht, Ralf

2014-08-01

In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Reuse of imputed data in microarray analysis increases imputation efficiency

PubMed Central

Kim, Ki-Yeol; Kim, Byoung-Jin; Yi, Gwan-Su

2004-01-01

Background The imputation of missing values is necessary for the efficient use of DNA microarray data, because many clustering algorithms and some statistical analysis require a complete data set. A few imputation methods for DNA microarray data have been introduced, but the efficiency of the methods was low and the validity of imputed values in these methods had not been fully checked. Results We developed a new cluster-based imputation method called sequential K-nearest neighbor (SKNN) method. This imputes the missing values sequentially from the gene having least missing values, and uses the imputed values for the later imputation. Although it uses the imputed values, the efficiency of this new method is greatly improved in its accuracy and computational complexity over the conventional KNN-based method and other methods based on maximum likelihood estimation. The performance of SKNN was in particular higher than other imputation methods for the data with high missing rates and large number of experiments. Application of Expectation Maximization (EM) to the SKNN method improved the accuracy, but increased computational time proportional to the number of iterations. The Multiple Imputation (MI) method, which is well known but not applied previously to microarray data, showed a similarly high accuracy as the SKNN method, with slightly higher dependency on the types of data sets. Conclusions Sequential reuse of imputed data in KNN-based imputation greatly increases the efficiency of imputation. The SKNN method should be practically useful to save the data of some microarray experiments which have high amounts of missing entries. The SKNN method generates reliable imputed values which can be used for further cluster-based analysis of microarray data. PMID:15504240

Advantages of RNA-seq compared to RNA microarrays for transcriptome profiling of anterior cruciate ligament tears.

PubMed

Rai, Muhammad Farooq; Tycksen, Eric D; Sandell, Linda J; Brophy, Robert H

2018-01-01

Microarrays and RNA-seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA-seq and microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA-seq and microarrays and to assess potential benefits of RNA-seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time-from-injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA-seq (N = 8). The correlation of biological replicates in RNA-seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarrays values were moderate. The cross-platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA-Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA-seq and microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484-497, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

An Advanced Approach to Simultaneous Monitoring of Multiple Bacteria in Space

NASA Technical Reports Server (NTRS)

Eggers, M.

1998-01-01

The utility of a novel microarray-based microbial analyzer was demonstrated by the rapid detection, imaging, and identification of a mixture of microorganisms found in a waste water sample from the Lunar-Mars Life Support Test Project through the synergistic combination of: (1) judicious RNA probe selection via algorithms developed by University of Houston scientists; (2) tuned surface chemistries developed by Baylor College of Medicine scientists to facilitate hybridization of rRNA targets to DNA probes under very low salt conditions, thereby minimizing secondary structure; and (3) integration of the microarray printing and detection/imaging instrumentation by Genometrix to complete the quantitative analysis of microorganism mixtures.

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

PubMed

Liu, Rui; Li, Wei; Cai, Tingting; Deng, Yang; Ding, Zhi; Liu, Yan; Zhu, Xuerui; Wang, Xin; Liu, Jie; Liang, Baowen; Zheng, Tiesong; Li, Jianlin

2018-05-02

A new aptamer microarray method on the TiO 2 -porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO 2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO 2 -PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B 1 (AFB 1 ), and 0.001-10 ng/mL for fumonisin B 1 (FB 1 ) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB 1 , and FB 1 , respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Surface plasmon resonance imaging system with Mach-Zehnder phase-shift interferometry for DNA micro-array hybridization

NASA Astrophysics Data System (ADS)

Hsiu, Feng-Ming; Chen, Shean-Jen; Tsai, Chien-Hung; Tsou, Chia-Yuan; Su, Y.-D.; Lin, G.-Y.; Huang, K.-T.; Chyou, Jin-Jung; Ku, Wei-Chih; Chiu, S.-K.; Tzeng, C.-M.

2002-09-01

Surface plasmon resonance (SPR) imaging system is presented as a novel technique based on modified Mach-Zehnder phase-shifting interferometry (PSI) for biomolecular interaction analysis (BIA), which measures the spatial phase variation of a resonantly reflected light in biomolecular interaction. In this technique, the micro-array SPR biosensors with over a thousand probe NDA spots can be detected simultaneously. Owing to the feasible and swift measurements, the micro-array SPR biosensors can be extensively applied to the nonspecific adsorption of protein, the membrane/protein interactions, and DNA hybridization. The detection sensitivity of the SPR PSI imaging system is improved to about 1 pg/mm2 for each spot over the conventional SPR imaging systems. The SPR PSI imaging system and its SPR sensors have been successfully used to observe slightly index change in consequence of argon gas flow through the nitrogen in real time, with high sensitivity, and at high-throughout screening rates.

Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants

PubMed Central

Walter, Andreas; Knapp, Brigitte A.; Farbmacher, Theresa; Ebner, Christian; Insam, Heribert; Franke‐Whittle, Ingrid H.

2012-01-01

Summary To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full‐scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real‐time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year. PMID:22950603

Tissue microarray immunohistochemical detection of brachyury is not a prognostic indicator in chordoma.

PubMed

Zhang, Linlin; Guo, Shang; Schwab, Joseph H; Nielsen, G Petur; Choy, Edwin; Ye, Shunan; Zhang, Zhan; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

2013-01-01

Brachyury is a marker for notochord-derived tissues and neoplasms, such as chordoma. However, the prognostic relevance of brachyury expression in chordoma is still unknown. The improvement of tissue microarray technology has provided the opportunity to perform analyses of tumor tissues on a large scale in a uniform and consistent manner. This study was designed with the use of tissue microarray to determine the expression of brachyury. Brachyury expression in chordoma tissues from 78 chordoma patients was analyzed by immunohistochemical staining of tissue microarray. The clinicopathologic parameters, including gender, age, location of tumor and metastatic status were evaluated. Fifty-nine of 78 (75.64%) tumors showed nuclear staining for brachyury, and among them, 29 tumors (49.15%) showed 1+ (<30% positive cells) staining, 15 tumors (25.42%) had 2+ (31% to 60% positive cells) staining, and 15 tumors (25.42%) demonstrated 3+ (61% to 100% positive cells) staining. Brachyury nuclear staining was detected more frequently in sacral chordomas than in chordomas of the mobile spine. However, there was no significant relationship between brachyury expression and other clinical variables. By Kaplan-Meier analysis, brachyury expression failed to produce any significant relationship with the overall survival rate. In conclusion, brachyury expression is not a prognostic indicator in chordoma.

Clinical application of chromosomal microarray analysis for the prenatal diagnosis of chromosomal abnormalities and copy number variations in fetuses with congenital heart disease.

PubMed

Xia, Yu; Yang, Yongchao; Huang, Shufang; Wu, Yueheng; Li, Ping; Zhuang, Jian

2018-03-24

This study aimed to determine chromosomal abnormalities and copy number variations (CNVs) in fetuses with congenital heart disease (CHD) by chromosomal microarray analysis (CMA). One hundred and ten cases with CHD detected by prenatal echocardiography were enrolled in the study; 27 cases were simple CHDs, and 83 were complex CHDs. Chromosomal microarray analysis was performed on the Affymetrix CytoScan HD platform. All annotated CNVs were validated by quantitative PCR. Chromosomal microarray analysis identified 6 cases with chromosomal abnormalities, including 2 cases with trisomy 21, 2 cases with trisomy 18, 1 case with trisomy 13, and 1 unusual case of mosaic trisomy 21. Pathogenic CNVs were detected in 15.5% (17/110) of the fetuses with CHDs, including 13 cases with CHD-associated CNVs. We further identified 10 genes as likely novel CHD candidate genes through gene functional enrichment analysis. We also found that pathogenic CMA results impacted the rate of pregnancy termination. This study shows that CMA is particularly effective for identifying chromosomal abnormalities and CNVs in fetuses with CHDs as well as having an effect on obstetrical outcomes. The elucidation of the genetic basis of CHDs will continue to expand our understanding of the etiology of CHDs. © 2018 John Wiley & Sons, Ltd.

Classification of Microarray Data Using Kernel Fuzzy Inference System

PubMed Central

Kumar Rath, Santanu

2014-01-01

The DNA microarray classification technique has gained more popularity in both research and practice. In real data analysis, such as microarray data, the dataset contains a huge number of insignificant and irrelevant features that tend to lose useful information. Classes with high relevance and feature sets with high significance are generally referred for the selected features, which determine the samples classification into their respective classes. In this paper, kernel fuzzy inference system (K-FIS) algorithm is applied to classify the microarray data (leukemia) using t-test as a feature selection method. Kernel functions are used to map original data points into a higher-dimensional (possibly infinite-dimensional) feature space defined by a (usually nonlinear) function ϕ through a mathematical process called the kernel trick. This paper also presents a comparative study for classification using K-FIS along with support vector machine (SVM) for different set of features (genes). Performance parameters available in the literature such as precision, recall, specificity, F-measure, ROC curve, and accuracy are considered to analyze the efficiency of the classification model. From the proposed approach, it is apparent that K-FIS model obtains similar results when compared with SVM model. This is an indication that the proposed approach relies on kernel function. PMID:27433543

Automated methods for multiplexed pathogen detection.

PubMed

Straub, Timothy M; Dockendorff, Brian P; Quiñonez-Díaz, Maria D; Valdez, Catherine O; Shutthanandan, Janani I; Tarasevich, Barbara J; Grate, Jay W; Bruckner-Lea, Cynthia J

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.

Automated Methods for Multiplexed Pathogen Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cyclermore » where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.« less

Biclustering of gene expression data using reactive greedy randomized adaptive search procedure.

PubMed

Dharan, Smitha; Nair, Achuthsankar S

2009-01-30

Biclustering algorithms belong to a distinct class of clustering algorithms that perform simultaneous clustering of both rows and columns of the gene expression matrix and can be a very useful analysis tool when some genes have multiple functions and experimental conditions are diverse. Cheng and Church have introduced a measure called mean squared residue score to evaluate the quality of a bicluster and has become one of the most popular measures to search for biclusters. In this paper, we review basic concepts of the metaheuristics Greedy Randomized Adaptive Search Procedure (GRASP)-construction and local search phases and propose a new method which is a variant of GRASP called Reactive Greedy Randomized Adaptive Search Procedure (Reactive GRASP) to detect significant biclusters from large microarray datasets. The method has two major steps. First, high quality bicluster seeds are generated by means of k-means clustering. In the second step, these seeds are grown using the Reactive GRASP, in which the basic parameter that defines the restrictiveness of the candidate list is self-adjusted, depending on the quality of the solutions found previously. We performed statistical and biological validations of the biclusters obtained and evaluated the method against the results of basic GRASP and as well as with the classic work of Cheng and Church. The experimental results indicate that the Reactive GRASP approach outperforms the basic GRASP algorithm and Cheng and Church approach. The Reactive GRASP approach for the detection of significant biclusters is robust and does not require calibration efforts.

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

PubMed Central

Prins, Theo W; van Dijk, Jeroen P; Beenen, Henriek G; Van Hoef, AM Angeline; Voorhuijzen, Marleen M; Schoen, Cor D; Aarts, Henk JM; Kok, Esther J

2008-01-01

Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected. PMID:19055784

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction.

PubMed

Prins, Theo W; van Dijk, Jeroen P; Beenen, Henriek G; Van Hoef, Am Angeline; Voorhuijzen, Marleen M; Schoen, Cor D; Aarts, Henk J M; Kok, Esther J

2008-12-04

To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation.In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected.

LITERATURE REVIEW OF MOLECULAR METHODS FOR SIMULTANEOUS DETECTION OF PATHOGENS IN WATER

EPA Science Inventory

This literature search is a review of molecular technologies (qPCR, microarray, microfluidics and lab-on-a-chip) for simultaneous detection of multiple waterborne pathogens in order to understand the state of the technology. The search content focuses on: pathogen detection witho...

Mining microarrays for metabolic meaning: nutritional regulation of hypothalamic gene expression.

PubMed

Mobbs, Charles V; Yen, Kelvin; Mastaitis, Jason; Nguyen, Ha; Watson, Elizabeth; Wurmbach, Elisa; Sealfon, Stuart C; Brooks, Andrew; Salton, Stephen R J

2004-06-01

DNA microarray analysis has been used to investigate relative changes in the level of gene expression in the CNS, including changes that are associated with disease, injury, psychiatric disorders, drug exposure or withdrawal, and memory formation. We have used oligonucleotide microarrays to identify hypothalamic genes that respond to nutritional manipulation. In addition to commonly used microarray analysis based on criteria such as fold-regulation, we have also found that simply carrying out multiple t tests then sorting by P value constitutes a highly reliable method to detect true regulation, as assessed by real-time polymerase chain reaction (PCR), even for relatively low abundance genes or relatively low magnitude of regulation. Such analyses directly suggested novel mechanisms that mediate effects of nutritional state on neuroendocrine function and are being used to identify regulated gene products that may elucidate the metabolic pathology of obese ob/ob, lean Vgf-/Vgf-, and other models with profound metabolic impairments.

LS-CAP: an algorithm for identifying cytogenetic aberrations in hepatocellular carcinoma using microarray data.

PubMed

He, Xianmin; Wei, Qing; Sun, Meiqian; Fu, Xuping; Fan, Sichang; Li, Yao

2006-05-01

Biological techniques such as Array-Comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH) and affymetrix single nucleotide pleomorphism (SNP) array have been used to detect cytogenetic aberrations. However, on genomic scale, these techniques are labor intensive and time consuming. Comparative genomic microarray analysis (CGMA) has been used to identify cytogenetic changes in hepatocellular carcinoma (HCC) using gene expression microarray data. However, CGMA algorithm can not give precise localization of aberrations, fails to identify small cytogenetic changes, and exhibits false negatives and positives. Locally un-weighted smoothing cytogenetic aberrations prediction (LS-CAP) based on local smoothing and binomial distribution can be expected to address these problems. LS-CAP algorithm was built and used on HCC microarray profiles. Eighteen cytogenetic abnormalities were identified, among them 5 were reported previously, and 12 were proven by CGH studies. LS-CAP effectively reduced the false negatives and positives, and precisely located small fragments with cytogenetic aberrations.

Micro-array isolation of circulating tumor cells (CTCs): the droplet biopsy chip

NASA Astrophysics Data System (ADS)

Panchapakesan, B.

2017-08-01

We present a new method for circulating tumor cell capture based on micro-array isolation from droplets. Called droplet biopsy, our technique uses a 76-element array of carbon nanotube devices functionalized with anti-EpCAM and antiHer2 antibodies for immunocapture of spiked breast cancer cells in the blood. This droplet biopsy chip can enable capture of CTCs based on both positive and negative selection strategy. Negative selection is achieved through depletion of contaminating leukocytes through the differential settling of blood into layers. We report 55%-100% cancer cell capture yield in this first droplet biopsy chip study. The droplet biopsy is an enabling idea where one can capture CTCs based on multiple biomarkers in a single blood sample.

Evolutionary Approach for Relative Gene Expression Algorithms

PubMed Central

Czajkowski, Marcin

2014-01-01

A Relative Expression Analysis (RXA) uses ordering relationships in a small collection of genes and is successfully applied to classiffication using microarray data. As checking all possible subsets of genes is computationally infeasible, the RXA algorithms require feature selection and multiple restrictive assumptions. Our main contribution is a specialized evolutionary algorithm (EA) for top-scoring pairs called EvoTSP which allows finding more advanced gene relations. We managed to unify the major variants of relative expression algorithms through EA and introduce weights to the top-scoring pairs. Experimental validation of EvoTSP on public available microarray datasets showed that the proposed solution significantly outperforms in terms of accuracy other relative expression algorithms and allows exploring much larger solution space. PMID:24790574

Feature Genes Selection Using Supervised Locally Linear Embedding and Correlation Coefficient for Microarray Classification

PubMed Central

Wang, Yun; Huang, Fangzhou

2018-01-01

The selection of feature genes with high recognition ability from the gene expression profiles has gained great significance in biology. However, most of the existing methods have a high time complexity and poor classification performance. Motivated by this, an effective feature selection method, called supervised locally linear embedding and Spearman's rank correlation coefficient (SLLE-SC2), is proposed which is based on the concept of locally linear embedding and correlation coefficient algorithms. Supervised locally linear embedding takes into account class label information and improves the classification performance. Furthermore, Spearman's rank correlation coefficient is used to remove the coexpression genes. The experiment results obtained on four public tumor microarray datasets illustrate that our method is valid and feasible. PMID:29666661

Feature Genes Selection Using Supervised Locally Linear Embedding and Correlation Coefficient for Microarray Classification.

PubMed

Xu, Jiucheng; Mu, Huiyu; Wang, Yun; Huang, Fangzhou

2018-01-01

The selection of feature genes with high recognition ability from the gene expression profiles has gained great significance in biology. However, most of the existing methods have a high time complexity and poor classification performance. Motivated by this, an effective feature selection method, called supervised locally linear embedding and Spearman's rank correlation coefficient (SLLE-SC 2 ), is proposed which is based on the concept of locally linear embedding and correlation coefficient algorithms. Supervised locally linear embedding takes into account class label information and improves the classification performance. Furthermore, Spearman's rank correlation coefficient is used to remove the coexpression genes. The experiment results obtained on four public tumor microarray datasets illustrate that our method is valid and feasible.

Ischemic and Nephrotoxic Acute Renal Failure are Distinguished by their Broad Transcriptomic Responses (102/160 char)

PubMed Central

Yuen, Peter S.T.; Jo, Sang-Kyung; Holly, Mikaela K.; Hu, Xuzhen; Star, Robert A.

2006-01-01

Acute renal failure (ARF) has a high morbidity and mortality. In animal ARF models, effective treatments must be administered before or shortly after the insult, limiting their clinical potential. We used microarrays to identify early biomarkers that distinguish ischemic from nephrotoxic ARF, or biomarkers that detect both injury types. We compared rat kidney transcriptomes 2 and 8 hours after ischemia/reperfusion and after mercuric chloride. Quality control and statistical analyses were necessary to normalize microarrays from different lots, eliminate outliers, and exclude unaltered genes. Principal component analysis revealed distinct ischemic and nephrotoxic trajectories, and clear array groupings. Therefore, we used supervised analysis, t-tests and fold changes, to compile gene lists for each group, exclusive or non-exclusive, alone or in combination. There was little network connectivity, even in the largest group. Some microarray-identified genes were validated by TaqMan assay, ruling out artifacts. Western blotting confirmed that HO-1 and ATF3 proteins were upregulated; however, unexpectedly, their localization changed within the kidney. HO-1 staining shifted from cortical (early) to outer stripe of the outer medulla (late), primarily in detaching cells, after mercuric chloride, but not ischemia/reperfusion. ATF3 staining was similar, but with additional early transient expression in the outer stripe after ischemia/reperfusion. We conclude that microarray-identified genes must be evaluated not only for protein levels, but also for anatomical distribution among different zones, nephron segments, or cell types. Although protein detection reagents are limited, microarray data lay a rich foundation to explore biomarkers, therapeutics, and pathophysiology of ARF. PMID:16507785

Resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses

PubMed Central

Wang, Zheng; Malanoski, Anthony P; Lin, Baochuan; Kidd, Carolyn; Long, Nina C; Blaney, Kate M; Thach, Dzung C; Tibbetts, Clark; Stenger, David A

2008-01-01

Background Febrile respiratory illness (FRI) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. A particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (HRV) and enteroviruses (HEV) which are frequent causes of FRI. Resequencing Pathogen Microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking. Results Using HRV and HEV as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. A minimal number of probe sequences (26 for HRV and 13 for HEV), which were potentially capable of detecting all serotypes of HRV and HEV, were determined and implemented on the Resequencing Pathogen Microarray RPM-Flu v.30/31 (Tessarae RPM-Flu). The specificities of designed probes were validated using 34 HRV and 28 HEV strains. All strains were successfully detected and identified at least to species level. 33 HRV strains and 16 HEV strains could be further differentiated to serotype level. Conclusion This study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse RNA viruses with a minimal number of prototype sequences. The results demonstrated that the newly designed RPM-Flu v.30/31 can provide comprehensive and specific analysis of HRV and HEV samples which implicates that this design strategy will be applicable for other genetically diverse viruses. PMID:19046445

Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

PubMed

Chen, Ming; Liu, Minghua; Yu, Lili; Cai, Guoru; Chen, Qinghai; Wu, Rong; Wang, Feng; Zhang, Bo; Jiang, Tianlun; Fu, Welling

2005-08-01

A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples.

Microarray characterization of gene expression changes in blood during acute ethanol exposure

PubMed Central

2013-01-01

Background As part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we performed a DNA microarray analysis of human whole blood samples from a five-time point study of subjects administered ethanol orally, followed by breathalyzer analysis, to monitor blood alcohol concentration (BAC) to discover significant gene expression changes in response to the ethanol exposure. Methods Subjects were administered either orange juice or orange juice with ethanol. Blood samples were taken based on BAC and total RNA was isolated from PaxGene™ blood tubes. The amplified cDNA was used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses to evaluate differential gene expression. Microarray data was analyzed in a pipeline fashion to summarize and normalize and the results evaluated for relative expression across time points with multiple methods. Candidate genes showing distinctive expression patterns in response to ethanol were clustered by pattern and further analyzed for related function, pathway membership and common transcription factor binding within and across clusters. RT-qPCR was used with representative genes to confirm relative transcript levels across time to those detected in microarrays. Results Microarray analysis of samples representing 0%, 0.04%, 0.08%, return to 0.04%, and 0.02% wt/vol BAC showed that changes in gene expression could be detected across the time course. The expression changes were verified by qRT-PCR. The candidate genes of interest (GOI) identified from the microarray analysis and clustered by expression pattern across the five BAC points showed seven coordinately expressed groups. Analysis showed function-based networks, shared transcription factor binding sites and signaling pathways for members of the clusters. These include hematological functions, innate immunity and inflammation functions, metabolic functions expected of ethanol metabolism, and pancreatic and hepatic function. Five of the seven clusters showed links to the p38 MAPK pathway. Conclusions The results of this study provide a first look at changing gene expression patterns in human blood during an acute rise in blood ethanol concentration and its depletion because of metabolism and excretion, and demonstrate that it is possible to detect changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study serves as a workflow to investigate the biology linked to expression changes across a time course and from these changes, to identify target genes that could serve as biomarkers linked to pilot performance. PMID:23883607

Profiling protein function with small molecule microarrays

PubMed Central

Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

2002-01-01

The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.

2006-10-01

The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. Themore » development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.« less

CRISPR Spacer Arrays for Detection of Viral Signatures from Acidic Hot Springs

NASA Astrophysics Data System (ADS)

Snyder, J. C.; Bateson, M. M.; Suciu, D.; Young, M. J.

2010-04-01

Viruses are the most abundant life-like entities on the planet Earth. Using CRISPR spacer sequences, we have developed a microarray-based approach to detecting viral signatures in the acidic hot springs of Yellowstone.

Development of a Native Fractionation Antigen Microarray for Autoantibody Profiling in Breast Cancer

DTIC Science & Technology

2011-10-01

Antigen Microarray for Autoantibody Profiling in Breast Cancer PRINCIPAL INVESTIGATOR: Brian C.-S. Liu, Ph.D...Profiling in Breast Cancer 5b. GRANT NUMBER W81XWH-09-1-0684 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Brian C.-S. Liu...NOTES 14. ABSTRACT The humoral response of a cancer patient may allow earlier detection of cancer than current methods allow. If so, the serum

Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery

PubMed Central

Karas, Vlad O; Sinnott-Armstrong, Nicholas A; Varghese, Vici; Shafer, Robert W; Greenleaf, William J; Sherlock, Gavin

2018-01-01

Abstract Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. PMID:29361139

Evaluation of three read-depth based CNV detection tools using whole-exome sequencing data.

PubMed

Yao, Ruen; Zhang, Cheng; Yu, Tingting; Li, Niu; Hu, Xuyun; Wang, Xiumin; Wang, Jian; Shen, Yiping

2017-01-01

Whole exome sequencing (WES) has been widely accepted as a robust and cost-effective approach for clinical genetic testing of small sequence variants. Detection of copy number variants (CNV) within WES data have become possible through the development of various algorithms and software programs that utilize read-depth as the main information. The aim of this study was to evaluate three commonly used, WES read-depth based CNV detection programs using high-resolution chromosomal microarray analysis (CMA) as a standard. Paired CMA and WES data were acquired for 45 samples. A total of 219 CNVs (size ranged from 2.3 kb - 35 mb) identified on three CMA platforms (Affymetrix, Agilent and Illumina) were used as standards. CNVs were called from WES data using XHMM, CoNIFER, and CNVnator with modified settings. All three software packages detected an elevated proportion of small variants (< 20 kb) compared to CMA. XHMM and CoNIFER had poor detection sensitivity (22.2 and 14.6%), which correlated with the number of capturing probes involved. CNVnator detected most variants and had better sensitivity (87.7%); however, suffered from an overwhelming detection of small CNVs below 20 kb, which required further confirmation. Size estimation of variants was exaggerated by CNVnator and understated by XHMM and CoNIFER. Low concordances of CNV, detected by three different read-depth based programs, indicate the immature status of WES-based CNV detection. Low sensitivity and uncertain specificity of WES-based CNV detection in comparison with CMA based CNV detection suggests that CMA will continue to play an important role in detecting clinical grade CNV in the NGS era, which is largely based on WES.

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

PubMed

Montagne, Louise; Derhourhi, Mehdi; Piton, Amélie; Toussaint, Bénédicte; Durand, Emmanuelle; Vaillant, Emmanuel; Thuillier, Dorothée; Gaget, Stefan; De Graeve, Franck; Rabearivelo, Iandry; Lansiaux, Amélie; Lenne, Bruno; Sukno, Sylvie; Desailloud, Rachel; Cnop, Miriam; Nicolescu, Ramona; Cohen, Lior; Zagury, Jean-François; Amouyal, Mélanie; Weill, Jacques; Muller, Jean; Sand, Olivier; Delobel, Bruno; Froguel, Philippe; Bonnefond, Amélie

2018-05-16

The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (n total  = 145). CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Assessing differential expression in two-color microarrays: a resampling-based empirical Bayes approach.

PubMed

Li, Dongmei; Le Pape, Marc A; Parikh, Nisha I; Chen, Will X; Dye, Timothy D

2013-01-01

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth's ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth's parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth's parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.

Differential prioritization between relevance and redundancy in correlation-based feature selection techniques for multiclass gene expression data.

PubMed

Ooi, Chia Huey; Chetty, Madhu; Teng, Shyh Wei

2006-06-23

Due to the large number of genes in a typical microarray dataset, feature selection looks set to play an important role in reducing noise and computational cost in gene expression-based tissue classification while improving accuracy at the same time. Surprisingly, this does not appear to be the case for all multiclass microarray datasets. The reason is that many feature selection techniques applied on microarray datasets are either rank-based and hence do not take into account correlations between genes, or are wrapper-based, which require high computational cost, and often yield difficult-to-reproduce results. In studies where correlations between genes are considered, attempts to establish the merit of the proposed techniques are hampered by evaluation procedures which are less than meticulous, resulting in overly optimistic estimates of accuracy. We present two realistically evaluated correlation-based feature selection techniques which incorporate, in addition to the two existing criteria involved in forming a predictor set (relevance and redundancy), a third criterion called the degree of differential prioritization (DDP). DDP functions as a parameter to strike the balance between relevance and redundancy, providing our techniques with the novel ability to differentially prioritize the optimization of relevance against redundancy (and vice versa). This ability proves useful in producing optimal classification accuracy while using reasonably small predictor set sizes for nine well-known multiclass microarray datasets. For multiclass microarray datasets, especially the GCM and NCI60 datasets, DDP enables our filter-based techniques to produce accuracies better than those reported in previous studies which employed similarly realistic evaluation procedures.

CAFE: an R package for the detection of gross chromosomal abnormalities from gene expression microarray data.

PubMed

Bollen, Sander; Leddin, Mathias; Andrade-Navarro, Miguel A; Mah, Nancy

2014-05-15

The current methods available to detect chromosomal abnormalities from DNA microarray expression data are cumbersome and inflexible. CAFE has been developed to alleviate these issues. It is implemented as an R package that analyzes Affymetrix *.CEL files and comes with flexible plotting functions, easing visualization of chromosomal abnormalities. CAFE is available from https://bitbucket.org/cob87icW6z/cafe/ as both source and compiled packages for Linux and Windows. It is released under the GPL version 3 license. CAFE will also be freely available from Bioconductor. sander.h.bollen@gmail.com or nancy.mah@mdc-berlin.de Supplementary data are available at Bioinformatics online.

[Oligonucleotide microarray for subtyping avian influenza virus].

PubMed

Xueqing, Han; Xiangmei, Lin; Yihong, Hou; Shaoqiang, Wu; Jian, Liu; Lin, Mei; Guangle, Jia; Zexiao, Yang

2008-09-01

Avian influenza viruses are important human and animal respiratory pathogens and rapid diagnosis of novel emerging avian influenza viruses is vital for effective global influenza surveillance. We developed an oligonucleotide microarray-based method for subtyping all avian influenza virus (16 HA and 9 NA subtypes). In total 25 pairs of primers specific for different subtypes and 1 pair of universal primers were carefully designed based on the genomic sequences of influenza A viruses retrieved from GenBank database. Several multiplex RT-PCR methods were then developed, and the target cDNAs of 25 subtype viruses were amplified by RT-PCR or overlapping PCR for evaluating the microarray. Further 52 oligonucleotide probes specific for all 25 subtype viruses were designed according to published gene sequences of avian influenza viruses in amplified target cDNAs domains, and a microarray for subtyping influenza A virus was developed. Then its specificity and sensitivity were validated by using different subtype strains and 2653 samples from 49 different areas. The results showed that all the subtypes of influenza virus could be identified simultaneously on this microarray with high sensitivity, which could reach to 2.47 pfu/mL virus or 2.5 ng target DNA. Furthermore, there was no cross reaction with other avian respiratory virus. An oligonucleotide microarray-based strategy for detection of avian influenza viruses has been developed. Such a diagnostic microarray will be useful in discovering and identifying all subtypes of avian influenza virus.

DNA Microarray for Detection of Macrolide Resistance Genes

PubMed Central

Cassone, Marco; D'Andrea, Marco M.; Iannelli, Francesco; Oggioni, Marco R.; Rossolini, Gian Maria; Pozzi, Gianni

2006-01-01

A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria. PMID:16723563

Characterization of genetic variability of Venezuelan equine encephalitis viruses

DOE PAGES

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.; ...

2016-04-07

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

On-Chip, Amplification-Free Quantification of Nucleic Acid for Point-of-Care Diagnosis

NASA Astrophysics Data System (ADS)

Yen, Tony Minghung

This dissertation demonstrates three physical device concepts to overcome limitations in point-of-care quantification of nucleic acids. Enabling sensitive, high throughput nucleic acid quantification on a chip, outside of hospital and centralized laboratory setting, is crucial for improving pathogen detection and cancer diagnosis and prognosis. Among existing platforms, microarray have the advantages of being amplification free, low instrument cost, and high throughput, but are generally less sensitive compared to sequencing and PCR assays. To bridge this performance gap, this dissertation presents theoretical and experimental progress to develop a platform nucleic acid quantification technology that is drastically more sensitive than current microarrays while compatible with microarray architecture. The first device concept explores on-chip nucleic acid enrichment by natural evaporation of nucleic acid solution droplet. Using a micro-patterned super-hydrophobic black silicon array device, evaporative enrichment is coupled with nano-liter droplet self-assembly workflow to produce a 50 aM concentration sensitivity, 6 orders of dynamic range, and rapid hybridization time at under 5 minutes. The second device concept focuses on improving target copy number sensitivity, instead of concentration sensitivity. A comprehensive microarray physical model taking into account of molecular transport, electrostatic intermolecular interactions, and reaction kinetics is considered to guide device optimization. Device pattern size and target copy number are optimized based on model prediction to achieve maximal hybridization efficiency. At a 100-mum pattern size, a quantum leap in detection limit of 570 copies is achieved using black silicon array device with self-assembled pico-liter droplet workflow. Despite its merits, evaporative enrichment on black silicon device suffers from coffee-ring effect at 100-mum pattern size, and thus not compatible with clinical patient samples. The third device concept utilizes an integrated optomechanical laser system and a Cytop microarray device to reverse coffee-ring effect during evaporative enrichment at 100-mum pattern size. This method, named "laser-induced differential evaporation" is expected to enable 570 copies detection limit for clinical samples in near future. While the work is ongoing as of the writing of this dissertation, a clear research plan is in place to implement this method on microarray platform toward clinical sample testing for disease applications and future commercialization.

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Leukemia and colon tumor detection based on microarray data classification using momentum backpropagation and genetic algorithm as a feature selection method

NASA Astrophysics Data System (ADS)

Wisesty, Untari N.; Warastri, Riris S.; Puspitasari, Shinta Y.

2018-03-01

Cancer is one of the major causes of mordibility and mortality problems in the worldwide. Therefore, the need of a system that can analyze and identify a person suffering from a cancer by using microarray data derived from the patient’s Deoxyribonucleic Acid (DNA). But on microarray data has thousands of attributes, thus making the challenges in data processing. This is often referred to as the curse of dimensionality. Therefore, in this study built a system capable of detecting a patient whether contracted cancer or not. The algorithm used is Genetic Algorithm as feature selection and Momentum Backpropagation Neural Network as a classification method, with data used from the Kent Ridge Bio-medical Dataset. Based on system testing that has been done, the system can detect Leukemia and Colon Tumor with best accuracy equal to 98.33% for colon tumor data and 100% for leukimia data. Genetic Algorithm as feature selection algorithm can improve system accuracy, which is from 64.52% to 98.33% for colon tumor data and 65.28% to 100% for leukemia data, and the use of momentum parameters can accelerate the convergence of the system in the training process of Neural Network.

Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

PubMed

Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

2015-10-01

The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of dkgB-linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica

PubMed Central

Guard, Jean; Sanchez-Ingunza, Roxana; Morales, Cesar; Stewart, Tod; Liljebjelke, Karen; Kessel, JoAnn; Ingram, Kim; Jones, Deana; Jackson, Charlene; Fedorka-Cray, Paula; Frye, Jonathan; Gast, Richard; Hinton, Arthur

2012-01-01

Two DNA-based methods were compared for the ability to assign serotype to 139 isolates of Salmonella enterica ssp. I. Intergenic sequence ribotyping (ISR) evaluated single nucleotide polymorphisms occurring in a 5S ribosomal gene region and flanking sequences bordering the gene dkgB. A DNA microarray hybridization method that assessed the presence and the absence of sets of genes was the second method. Serotype was assigned for 128 (92.1%) of submissions by the two DNA methods. ISR detected mixtures of serotypes within single colonies and it cost substantially less than Kauffmann–White serotyping and DNA microarray hybridization. Decreasing the cost of serotyping S. enterica while maintaining reliability may encourage routine testing and research. PMID:22998607

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

PubMed

Chruscinski, Andrzej; Huang, Flora Y Y; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C; Kozuszko, Stella; Tinckam, Kathryn J; Rao, Vivek; Dunn, Shannon E; Persinger, Michael A; Levy, Gary A; Ross, Heather J

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation

PubMed Central

Chruscinski, Andrzej; Huang, Flora Y. Y.; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C.; Kozuszko, Stella; Tinckam, Kathryn J.; Rao, Vivek; Dunn, Shannon E.; Persinger, Michael A.; Levy, Gary A.; Ross, Heather J.

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR. PMID:26967734

An object model and database for functional genomics.

PubMed

Jones, Andrew; Hunt, Ela; Wastling, Jonathan M; Pizarro, Angel; Stoeckert, Christian J

2004-07-10

Large-scale functional genomics analysis is now feasible and presents significant challenges in data analysis, storage and querying. Data standards are required to enable the development of public data repositories and to improve data sharing. There is an established data format for microarrays (microarray gene expression markup language, MAGE-ML) and a draft standard for proteomics (PEDRo). We believe that all types of functional genomics experiments should be annotated in a consistent manner, and we hope to open up new ways of comparing multiple datasets used in functional genomics. We have created a functional genomics experiment object model (FGE-OM), developed from the microarray model, MAGE-OM and two models for proteomics, PEDRo and our own model (Gla-PSI-Glasgow Proposal for the Proteomics Standards Initiative). FGE-OM comprises three namespaces representing (i) the parts of the model common to all functional genomics experiments; (ii) microarray-specific components; and (iii) proteomics-specific components. We believe that FGE-OM should initiate discussion about the contents and structure of the next version of MAGE and the future of proteomics standards. A prototype database called RNA And Protein Abundance Database (RAPAD), based on FGE-OM, has been implemented and populated with data from microbial pathogenesis. FGE-OM and the RAPAD schema are available from http://www.gusdb.org/fge.html, along with a set of more detailed diagrams. RAPAD can be accessed by registration at the site.

Quantitative comparison of microarray experiments with published leukemia related gene expression signatures.

PubMed

Klein, Hans-Ulrich; Ruckert, Christian; Kohlmann, Alexander; Bullinger, Lars; Thiede, Christian; Haferlach, Torsten; Dugas, Martin

2009-12-15

Multiple gene expression signatures derived from microarray experiments have been published in the field of leukemia research. A comparison of these signatures with results from new experiments is useful for verification as well as for interpretation of the results obtained. Currently, the percentage of overlapping genes is frequently used to compare published gene signatures against a signature derived from a new experiment. However, it has been shown that the percentage of overlapping genes is of limited use for comparing two experiments due to the variability of gene signatures caused by different array platforms or assay-specific influencing parameters. Here, we present a robust approach for a systematic and quantitative comparison of published gene expression signatures with an exemplary query dataset. A database storing 138 leukemia-related published gene signatures was designed. Each gene signature was manually annotated with terms according to a leukemia-specific taxonomy. Two analysis steps are implemented to compare a new microarray dataset with the results from previous experiments stored and curated in the database. First, the global test method is applied to assess gene signatures and to constitute a ranking among them. In a subsequent analysis step, the focus is shifted from single gene signatures to chromosomal aberrations or molecular mutations as modeled in the taxonomy. Potentially interesting disease characteristics are detected based on the ranking of gene signatures associated with these aberrations stored in the database. Two example analyses are presented. An implementation of the approach is freely available as web-based application. The presented approach helps researchers to systematically integrate the knowledge derived from numerous microarray experiments into the analysis of a new dataset. By means of example leukemia datasets we demonstrate that this approach detects related experiments as well as related molecular mutations and may help to interpret new microarray data.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

PubMed

Jaakson, K; Zernant, J; Külm, M; Hutchinson, A; Tonisson, N; Glavac, D; Ravnik-Glavac, M; Hawlina, M; Meltzer, M R; Caruso, R C; Testa, F; Maugeri, A; Hoyng, C B; Gouras, P; Simonelli, F; Lewis, R A; Lupski, J R; Cremers, F P M; Allikmets, R

2003-11-01

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research. Copyright 2003 Wiley-Liss, Inc.

Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina Acetivorans Grown with Acetate Versus Methanol*

PubMed Central

Li, Lingyun; Li, Qingbo; Rohlin, Lars; Kim, UnMi; Salmon, Kirsty; Rejtar, Tomas; Gunsalus, Robert P.; Karger, Barry L.; Ferry, James G.

2008-01-01

Summary Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs. methanol-grown cells metabolically labeled with 14N vs. 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs. methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥ 3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥ 2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs. methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate. PMID:17269732

Characterization of Antimicrobial Resistance Patterns and Detection of Virulence Genes in Campylobacter Isolates in Italy

PubMed Central

Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-01-01

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations. PMID:24556669

Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy.

PubMed

Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-02-19

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

A low density microarray method for the identification of human papillomavirus type 18 variants.

PubMed

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C

2013-09-26

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

PubMed Central

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

Sensitive detection of major food allergens in breast milk: first gateway for allergenic contact during breastfeeding.

PubMed

Pastor-Vargas, C; Maroto, A S; Díaz-Perales, A; Villaba, M; Casillas Diaz, N; Vivanco, F; Cuesta-Herranz, J

2015-08-01

Food allergy is recognized as a major public health issue, especially in early childhood. It has been hypothesized that early sensitization to food allergens maybe due to their ingestion as components dissolved in the milk during the breastfeeding, explaining reaction to a food, which has never been taken before. Thus, the aim of this work has been to detect the presence of the food allergens in breast milk by microarray technology. We produced a homemade microarray with antibodies produced against major food allergens. The antibody microarray was incubated with breast milk from 14 women collected from Fundación Jiménez Díaz Hospital. In this way, we demonstrated the presence of major foods allergens in breast milk. The analysis of allergens presented in breast milk could be a useful tool in allergy prevention and could provide us a key data on the role of this feeding in tolerance induction or sensitization in children. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies.

PubMed

Martín, Verónica; Perales, Celia; Fernández-Algar, María; Dos Santos, Helena G; Garrido, Patricia; Pernas, María; Parro, Víctor; Moreno, Miguel; García-Pérez, Javier; Alcamí, José; Torán, José Luis; Abia, David; Domingo, Esteban; Briones, Carlos

2016-01-01

The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.

Short non-coding RNAs as bacteria species identifiers detected by surface plasmon resonance enhanced common path interferometry

NASA Astrophysics Data System (ADS)

Greef, Charles; Petropavlovskikh, Viatcheslav; Nilsen, Oyvind; Khattatov, Boris; Plam, Mikhail; Gardner, Patrick; Hall, John

2008-04-01

Small non-coding RNA sequences have recently been discovered as unique identifiers of certain bacterial species, raising the possibility that they can be used as highly specific Biowarfare Agent detection markers in automated field deployable integrated detection systems. Because they are present in high abundance they could allow genomic based bacterial species identification without the need for pre-assay amplification. Further, a direct detection method would obviate the need for chemical labeling, enabling a rapid, efficient, high sensitivity mechanism for bacterial detection. Surface Plasmon Resonance enhanced Common Path Interferometry (SPR-CPI) is a potentially market disruptive, high sensitivity dual technology that allows real-time direct multiplex measurement of biomolecule interactions, including small molecules, nucleic acids, proteins, and microbes. SPR-CPI measures differences in phase shift of reflected S and P polarized light under Total Internal Reflection (TIR) conditions at a surface, caused by changes in refractive index induced by biomolecular interactions within the evanescent field at the TIR interface. The measurement is performed on a microarray of discrete 2-dimensional areas functionalized with biomolecule capture reagents, allowing simultaneous measurement of up to 100 separate analytes. The optical beam encompasses the entire microarray, allowing a solid state detector system with no scanning requirement. Output consists of simultaneous voltage measurements proportional to the phase differences resulting from the refractive index changes from each microarray feature, and is automatically processed and displayed graphically or delivered to a decision making algorithm, enabling a fully automatic detection system capable of rapid detection and quantification of small nucleic acids at extremely sensitive levels. Proof-of-concept experiments on model systems and cell culture samples have demonstrated utility of the system, and efforts are in progress for full development and deployment of the device. The technology has broad applicability as a universal detection platform for BWA detection, medical diagnostics, and drug discovery research, and represents a new class of instrumentation as a rapid, high sensitivity, label-free methodology.

Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

PubMed Central

Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

2012-01-01

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data

PubMed Central

Watson, Christopher M.; Crinnion, Laura A.; Gurgel‐Gianetti, Juliana; Harrison, Sally M.; Daly, Catherine; Antanavicuite, Agne; Lascelles, Carolina; Markham, Alexander F.; Pena, Sergio D. J.; Bonthron, David T.

2015-01-01

ABSTRACT Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease‐causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome‐wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution. PMID:26037133

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

MiMiR – an integrated platform for microarray data sharing, mining and analysis

PubMed Central

Tomlinson, Chris; Thimma, Manjula; Alexandrakis, Stelios; Castillo, Tito; Dennis, Jayne L; Brooks, Anthony; Bradley, Thomas; Turnbull, Carly; Blaveri, Ekaterini; Barton, Geraint; Chiba, Norie; Maratou, Klio; Soutter, Pat; Aitman, Tim; Game, Laurence

2008-01-01

Background Despite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Microarray data Mining Resource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data. Results A user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package. Conclusion The new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centre's large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies. PMID:18801157

MiMiR--an integrated platform for microarray data sharing, mining and analysis.

PubMed

Tomlinson, Chris; Thimma, Manjula; Alexandrakis, Stelios; Castillo, Tito; Dennis, Jayne L; Brooks, Anthony; Bradley, Thomas; Turnbull, Carly; Blaveri, Ekaterini; Barton, Geraint; Chiba, Norie; Maratou, Klio; Soutter, Pat; Aitman, Tim; Game, Laurence

2008-09-18

Despite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Microarray data Mining Resource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data. A user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package. The new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centre's large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies.

Chromosomal microarray in clinical diagnosis: a study of 337 patients with congenital anomalies and developmental delays or intellectual disability.

PubMed

Sansović, Ivona; Ivankov, Ana-Maria; Bobinec, Adriana; Kero, Mijana; Barišić, Ingeborg

2017-06-14

To determine the diagnostic yield and criteria that could help to classify and interpret the copy number variations (CNVs) detected by chromosomal microarray (CMA) technique in patients with congenital and developmental abnormalities including dysmorphia, developmental delay (DD) or intellectual disability (ID), autism spectrum disorders (ASD) and congenital anomalies (CA). CMA analysis was performed in 337 patients with DD/ID with or without dysmorphism, ASD, and/or CA. In 30 of 337 patients, chromosomal imbalances had previously been detected by classical cytogenetic and molecular cytogenetic methods. In 73 of 337 patients, clinically relevant variants were detected and better characterized. Most of them were >1 Mb. Variants of unknown clinical significance (VOUS) were discovered in 35 patients. The most common VOUS size category was <300 kb (40.5%). Deletions and de novo imbalances were more frequent in pathogenic CNV than in VOUS category. CMA had a high diagnostic yield of 43/307, excluding patients previously detected by other methods. CMA was valuable in establishing the diagnosis in a high proportion of patients. Criteria for classification and interpretation of CNVs include CNV size and type, mode of inheritance, and genotype-phenotype correlation. Agilent ISCA v2 Human Genome 8x60 K oligonucleotide microarray format proved to be reasonable resolution for clinical use, particularly in the regions that are recommended by the International Standard Cytogenomic Array (ISCA) Consortium and associated with well-established syndromes.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

PubMed

Adak, Avijit K; Li, Ben-Yuan; Huang, Li-De; Lin, Ting-Wei; Chang, Tsung-Che; Hwang, Kuo Chu; Lin, Chun-Cheng

2014-07-09

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Inkjet-assisted layer-by-layer printing of quantum dot/enzyme microarrays for highly sensitive detection of organophosphorous pesticides.

PubMed

Luan, Enxiao; Zheng, Zhaozhu; Li, Xinyu; Gu, Hongxi; Liu, Shaoqin

2016-04-15

We present a facile fabrication of layer-by-layer (LbL) microarrays of quantum dots (QDs) and acetylcholinesterase enzyme (AChE). The resulting arrays had several unique properties, such as low cost, high integration and excellent flexibility and time-saving. The presence of organophosphorous pesticides (OPs) can inhibit the AChE activity and thus changes the fluorescent intensity of QDs/AChE microscopic dot arrays. Therefore, the QDs/AChE microscopic dot arrays were used for the sensitive visual detection of OPs. Linear calibration for parathion and paraoxon was obtained in the range of 5-100 μg L(-1) under the optimized conditions with the limit of detection (LOD) of 10 μg L(-1). The arrays have been successfully used for detection of OPs in fruits and water real samples. The new array was validated by comparison with conventional high performance liquid chromatography-mass spectrometry (HPLC-MS). Copyright © 2016 Elsevier B.V. All rights reserved.

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

PubMed

Consolandi, Clarissa

2009-01-01

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

NAIMA as a solution for future GMO diagnostics challenges.

PubMed

Dobnik, David; Morisset, Dany; Gruden, Kristina

2010-03-01

In the field of genetically modified organism (GMO) diagnostics, real-time PCR has been the method of choice for target detection and quantification in most laboratories. Despite its numerous advantages, however, the lack of a true multiplexing option may render real-time PCR less practical in the face of future GMO detection challenges such as the multiplicity and increasing complexity of new transgenic events, as well as the repeated occurrence of unauthorized GMOs on the market. In this context, we recently reported the development of a novel multiplex quantitative DNA-based target amplification method, named NASBA implemented microarray analysis (NAIMA), which is suitable for sensitive, specific and quantitative detection of GMOs on a microarray. In this article, the performance of NAIMA is compared with that of real-time PCR, the focus being their performances in view of the upcoming challenge to detect/quantify an increasing number of possible GMOs at a sustainable cost and affordable staff effort. Finally, we present our conclusions concerning the applicability of NAIMA for future use in GMO diagnostics.

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

PubMed Central

Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

2005-01-01

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

ArrayWiki: an enabling technology for sharing public microarray data repositories and meta-analyses

PubMed Central

Stokes, Todd H; Torrance, JT; Li, Henry; Wang, May D

2008-01-01

Background A survey of microarray databases reveals that most of the repository contents and data models are heterogeneous (i.e., data obtained from different chip manufacturers), and that the repositories provide only basic biological keywords linking to PubMed. As a result, it is difficult to find datasets using research context or analysis parameters information beyond a few keywords. For example, to reduce the "curse-of-dimension" problem in microarray analysis, the number of samples is often increased by merging array data from different datasets. Knowing chip data parameters such as pre-processing steps (e.g., normalization, artefact removal, etc), and knowing any previous biological validation of the dataset is essential due to the heterogeneity of the data. However, most of the microarray repositories do not have meta-data information in the first place, and do not have a a mechanism to add or insert this information. Thus, there is a critical need to create "intelligent" microarray repositories that (1) enable update of meta-data with the raw array data, and (2) provide standardized archiving protocols to minimize bias from the raw data sources. Results To address the problems discussed, we have developed a community maintained system called ArrayWiki that unites disparate meta-data of microarray meta-experiments from multiple primary sources with four key features. First, ArrayWiki provides a user-friendly knowledge management interface in addition to a programmable interface using standards developed by Wikipedia. Second, ArrayWiki includes automated quality control processes (caCORRECT) and novel visualization methods (BioPNG, Gel Plots), which provide extra information about data quality unavailable in other microarray repositories. Third, it provides a user-curation capability through the familiar Wiki interface. Fourth, ArrayWiki provides users with simple text-based searches across all experiment meta-data, and exposes data to search engine crawlers (Semantic Agents) such as Google to further enhance data discovery. Conclusions Microarray data and meta information in ArrayWiki are distributed and visualized using a novel and compact data storage format, BioPNG. Also, they are open to the research community for curation, modification, and contribution. By making a small investment of time to learn the syntax and structure common to all sites running MediaWiki software, domain scientists and practioners can all contribute to make better use of microarray technologies in research and medical practices. ArrayWiki is available at . PMID:18541053

Biclustering of gene expression data using reactive greedy randomized adaptive search procedure

PubMed Central

Dharan, Smitha; Nair, Achuthsankar S

2009-01-01

Background Biclustering algorithms belong to a distinct class of clustering algorithms that perform simultaneous clustering of both rows and columns of the gene expression matrix and can be a very useful analysis tool when some genes have multiple functions and experimental conditions are diverse. Cheng and Church have introduced a measure called mean squared residue score to evaluate the quality of a bicluster and has become one of the most popular measures to search for biclusters. In this paper, we review basic concepts of the metaheuristics Greedy Randomized Adaptive Search Procedure (GRASP)-construction and local search phases and propose a new method which is a variant of GRASP called Reactive Greedy Randomized Adaptive Search Procedure (Reactive GRASP) to detect significant biclusters from large microarray datasets. The method has two major steps. First, high quality bicluster seeds are generated by means of k-means clustering. In the second step, these seeds are grown using the Reactive GRASP, in which the basic parameter that defines the restrictiveness of the candidate list is self-adjusted, depending on the quality of the solutions found previously. Results We performed statistical and biological validations of the biclusters obtained and evaluated the method against the results of basic GRASP and as well as with the classic work of Cheng and Church. The experimental results indicate that the Reactive GRASP approach outperforms the basic GRASP algorithm and Cheng and Church approach. Conclusion The Reactive GRASP approach for the detection of significant biclusters is robust and does not require calibration efforts. PMID:19208127

Clinical Performance of an Ultrahigh Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

PubMed

Ho, Karen S; Twede, Hope; Vanzo, Rena; Harward, Erin; Hensel, Charles H; Martin, Megan M; Page, Stephanie; Peiffer, Andreas; Mowery-Rushton, Patricia; Serrano, Moises; Wassman, E Robert

2016-01-01

Copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) significantly contribute to the etiology of neurodevelopmental disorders, such as developmental delay (DD), intellectual disability (ID), and autism spectrum disorder (ASD). This study summarizes the results of 3.5 years of CMA testing by a CLIA-certified clinical testing laboratory 5487 patients with neurodevelopmental conditions were clinically evaluated for rare copy number variants using a 2.8-million probe custom CMA optimized for the detection of CNVs associated with neurodevelopmental disorders. We report an overall detection rate of 29.4% in our neurodevelopmental cohort, which rises to nearly 33% when cases with DD/ID and/or MCA only are considered. The detection rate for the ASD cohort is also significant, at 25%. Additionally, we find that detection rate and pathogenic yield of CMA vary significantly depending on the primary indications for testing, the age of the individuals tested, and the specialty of the ordering doctor. We also report a significant difference between the detection rate on the ultrahigh resolution optimized array in comparison to the array from which it originated. This increase in detection can significantly contribute to the efficient and effective medical management of neurodevelopmental conditions in the clinic.

DEVELOPMENT AND EVALUATION OF A MICROARRAY APPROACH TO DETECT AND GENOTYPE NOROVIRUSES IN WATER

EPA Science Inventory

Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) base...

Detection of foodborne pathogens using microarray technology

USDA-ARS?s Scientific Manuscript database

Assays based on the polymerase chain reaction (PCR) are now accepted methods for rapidly confirming the presence or absence of specific pathogens in foods and other types of samples. Conventional PCR requires the use of agarose gel electrophoresis to detect the PCR product; whereas, real-time PCR c...

An automated multiplex specific IgE assay system using a photoimmobilized microarray.

PubMed

Ito, Yoshihiro; Moritsugu, Nozomi; Matsue, Takahisa; Mitsukoshi, Kiyomi; Ayame, Hirohito; Okochi, Norihiko; Hattori, Hideshi; Tashiro, Hideo; Sato, Sakura; Ebisawa, Motohiro

2012-11-15

An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 μL of serum within a period of 20 min. Copyright © 2012 Elsevier B.V. All rights reserved.

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH

PubMed Central

2009-01-01

Background Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient against a normal reference sample. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Instead, we apply an experimental loop design that compares three patients in three hybridizations. Results We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of 27 patients seen at our genetics center, we observed that the linear models of the log ratios are advantageous over the mixed models of the absolute intensities. Conclusion The loop design and the performance of the statistical analysis contribute to the quick adoption of array CGH as a routine diagnostic tool. They lower the detection limit of mosaicisms and improve the assignment of copy number variation for genetic association studies. PMID:19925645

Using microarray analysis to evaluate genetic polymorphisms involved in the metabolism of environmental chemicals.

PubMed

Ban, Susumu; Kondo, Tomoko; Ishizuka, Mayumi; Sasaki, Seiko; Konishi, Kanae; Washino, Noriaki; Fujita, Syoichi; Kishi, Reiko

2007-05-01

The field of molecular biology currently faces the need for a comprehensive method of evaluating individual differences derived from genetic variation in the form of single nucleotide polymorphisms (SNPs). SNPs in human genes are generally considered to be very useful in determining inherited genetic disorders, susceptibility to certain diseases, and cancer predisposition. Quick and accurate discrimination of SNPs is the key characteristic of technology used in DNA diagnostics. For this study, we first developed a DNA microarray and then evaluated its efficacy by determining the detection ability and validity of this method. Using DNA obtained from 380 pregnant Japanese women, we examined 13 polymorphisms of 9 genes, which are associated with the metabolism of environmental chemical compounds found in high frequency among Japanese populations. The ability to detect CYP1A1 I462V, CYP1B1 L432V, GSTP1 I105V and AhR R554K gene polymorphisms was above 98%, and agreement rates when compared with real time PCR analysis methods (kappa values) showed high validity: 0.98 (0.96), 0.97 (0.93), 0.90 (0.81), 0.90 (0.91), respectively. While this DNA microarray analysis should prove important as a method for initial screening, it is still necessary that we find better methods for improving the detection of other gene polymorphisms not part of this study.

Simultaneous subtyping and pathotyping of avian influenza viruses in chickens in Taiwan using reverse transcription loop-mediated isothermal amplification and microarray

PubMed Central

WANG, Lih-Chiann; HUANG, Dean; CHEN, Hui-Wen

2016-01-01

The H6N1 avian influenza virus has circulated in Taiwan for more than 40 years. The sporadic activity of low pathogenic H5N2 virus has been noted since 2003, and highly pathogenic H5N2 avian influenza virus has been detected since 2008. Ressortant viruses between H6N1 and H5N2 viruses have become established and enzootic in chickens throughout Taiwan. Outbreaks caused by Novel highly pathogenic H5 avian influenza viruses whose HA genes were closely related to that of the H5N8 virus isolated from ducks in Korea in 2014 were isolated from outbreaks in Taiwan since early 2015. The avian influenza virus infection status is becoming much more complicated in chickens in Taiwan. This necessitates a rapid and simple approach to detect and differentiate the viruses that prevail. H6N1, H5N2 and novel H5 viruses were simultaneously subtyped and pathotyped in this study using reverse transcription loop-mediated isothermal amplification and microarray, with detection limits of 10°, 101 and 10° viral copy numbers, respectively. The microarray signals were read by the naked eye with no expensive equipment needed. The method developed in this study could greatly improve avian influenza virus surveillance efficiency. PMID:27086860

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH.

PubMed

Allemeersch, Joke; Van Vooren, Steven; Hannes, Femke; De Moor, Bart; Vermeesch, Joris Robert; Moreau, Yves

2009-11-19

Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient against a normal reference sample. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Instead, we apply an experimental loop design that compares three patients in three hybridizations. We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of 27 patients seen at our genetics center, we observed that the linear models of the log ratios are advantageous over the mixed models of the absolute intensities. The loop design and the performance of the statistical analysis contribute to the quick adoption of array CGH as a routine diagnostic tool. They lower the detection limit of mosaicisms and improve the assignment of copy number variation for genetic association studies.

Digital microarray analysis for digital artifact genomics

NASA Astrophysics Data System (ADS)

Jaenisch, Holger; Handley, James; Williams, Deborah

2013-06-01

We implement a Spatial Voting (SV) based analogy of microarray analysis for digital gene marker identification in malware code sections. We examine a famous set of malware formally analyzed by Mandiant and code named Advanced Persistent Threat (APT1). APT1 is a Chinese organization formed with specific intent to infiltrate and exploit US resources. Manidant provided a detailed behavior and sting analysis report for the 288 malware samples available. We performed an independent analysis using a new alternative to the traditional dynamic analysis and static analysis we call Spatial Analysis (SA). We perform unsupervised SA on the APT1 originating malware code sections and report our findings. We also show the results of SA performed on some members of the families associated by Manidant. We conclude that SV based SA is a practical fast alternative to dynamics analysis and static analysis.

Detection of copy number variations in epilepsy using exome data.

PubMed

Tsuchida, N; Nakashima, M; Kato, M; Heyman, E; Inui, T; Haginoya, K; Watanabe, S; Chiyonobu, T; Morimoto, M; Ohta, M; Kumakura, A; Kubota, M; Kumagai, Y; Hamano, S-I; Lourenco, C M; Yahaya, N A; Ch'ng, G-S; Ngu, L-H; Fattal-Valevski, A; Weisz Hubshman, M; Orenstein, N; Marom, D; Cohen, L; Goldberg-Stern, H; Uchiyama, Y; Imagawa, E; Mizuguchi, T; Takata, A; Miyake, N; Nakajima, H; Saitsu, H; Miyatake, S; Matsumoto, N

2018-03-01

Epilepsies are common neurological disorders and genetic factors contribute to their pathogenesis. Copy number variations (CNVs) are increasingly recognized as an important etiology of many human diseases including epilepsy. Whole-exome sequencing (WES) is becoming a standard tool for detecting pathogenic mutations and has recently been applied to detecting CNVs. Here, we analyzed 294 families with epilepsy using WES, and focused on 168 families with no causative single nucleotide variants in known epilepsy-associated genes to further validate CNVs using 2 different CNV detection tools using WES data. We confirmed 18 pathogenic CNVs, and 2 deletions and 2 duplications at chr15q11.2 of clinically unknown significance. Of note, we were able to identify small CNVs less than 10 kb in size, which might be difficult to detect by conventional microarray. We revealed 2 cases with pathogenic CNVs that one of the 2 CNV detection tools failed to find, suggesting that using different CNV tools is recommended to increase diagnostic yield. Considering a relatively high discovery rate of CNVs (18 out of 168 families, 10.7%) and successful detection of CNV with <10 kb in size, CNV detection by WES may be able to surrogate, or at least complement, conventional microarray analysis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

PubMed

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

2015-10-06

Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

Segment and Fit Thresholding: A New Method for Image Analysis Applied to Microarray and Immunofluorescence Data

PubMed Central

Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M.; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E.; Allen, Peter J.; Sempere, Lorenzo F.; Haab, Brian B.

2016-01-01

Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis. PMID:26339978

R Script Approach to Infer Toxoplasma Infection Mechanisms From Microarrays and Domain-Domain Protein Interactions

PubMed Central

Arenas, Ailan F; Salcedo, Gladys E; Gomez-Marin, Jorge E

2017-01-01

Pathogen-host protein-protein interaction systems examine the interactions between the protein repertoires of 2 distinct organisms. Some of these pathogen proteins interact with the host protein system and may manipulate it for their own advantages. In this work, we designed an R script by concatenating 2 functions called rowDM and rowCVmed to infer pathogen-host interaction using previously reported microarray data, including host gene enrichment analysis and the crossing of interspecific domain-domain interactions. We applied this script to the Toxoplasma-host system to describe pathogen survival mechanisms from human, mouse, and Toxoplasma Gene Expression Omnibus series. Our outcomes exhibited similar results with previously reported microarray analyses, but we found other important proteins that could contribute to toxoplasma pathogenesis. We observed that Toxoplasma ROP38 is the most differentially expressed protein among toxoplasma strains. Enrichment analysis and KEGG mapping indicated that the human retinal genes most affected by Toxoplasma infections are those related to antiapoptotic mechanisms. We suggest that proteins PIK3R1, PRKCA, PRKCG, PRKCB, HRAS, and c-JUN could be the possible substrates for differentially expressed Toxoplasma kinase ROP38. Likewise, we propose that Toxoplasma causes overexpression of apoptotic suppression human genes. PMID:29317802

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarrays. 22-24 January 2001, Zurich, Switzerland.

PubMed

Jain, K K

2001-02-01

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarray technology covered the latest advances in this technology and applications in life sciences. Highlights of the meetings are reported briefly with emphasis on applications in genomics, drug discovery and molecular diagnostics. There was an emphasis on microfluidics because of the wide applications in laboratory and drug discovery. The lab-on-a-chip provides the facilities of a complete laboratory in a hand-held miniature device. Several microarray systems have been used for hybridisation and detection techniques. Oligonucleotide scanning arrays provide a versatile tool for the analysis of nucleic acid interactions and provide a platform for improving the array-based methods for investigation of antisense therapeutics. A method for analysing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional scanner has considerable potential in molecular diagnostics. Various applications of microarray technology for high-throughput screening in drug discovery and single nucleotide polymorphisms (SNP) analysis were discussed. Protein chips have important applications in proteomics. With the considerable amount of data generated by the different technologies using microarrays, it is obvious that the reading of the information and its interpretation and management through the use of bioinformatics is essential. Various techniques for data analysis were presented. Biochip and microarray technology has an essential role to play in the evolving trends in healthcare, which integrate diagnosis with prevention/treatment and emphasise personalised medicines.

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Analytical Protein Microarrays: Advancements Towards Clinical Applications

PubMed Central

Sauer, Ursula

2017-01-01

Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems. PMID:28146048

Bayesian hierarchical modeling for subject-level response classification in peptide microarray immunoassays

PubMed Central

Imholte, Gregory; Gottardo, Raphael

2017-01-01

Summary The peptide microarray immunoassay simultaneously screens sample serum against thousands of peptides, determining the presence of antibodies bound to array probes. Peptide microarrays tiling immunogenic regions of pathogens (e.g. envelope proteins of a virus) are an important high throughput tool for querying and mapping antibody binding. Because of the assay’s many steps, from probe synthesis to incubation, peptide microarray data can be noisy with extreme outliers. In addition, subjects may produce different antibody profiles in response to an identical vaccine stimulus or infection, due to variability among subjects’ immune systems. We present a robust Bayesian hierarchical model for peptide microarray experiments, pepBayes, to estimate the probability of antibody response for each subject/peptide combination. Heavy-tailed error distributions accommodate outliers and extreme responses, and tailored random effect terms automatically incorporate technical effects prevalent in the assay. We apply our model to two vaccine trial datasets to demonstrate model performance. Our approach enjoys high sensitivity and specificity when detecting vaccine induced antibody responses. A simulation study shows an adaptive thresholding classification method has appropriate false discovery rate control with high sensitivity, and receiver operating characteristics generated on vaccine trial data suggest that pepBayes clearly separates responses from non-responses. PMID:27061097

The laboratory-clinician team: a professional call to action to improve communication and collaboration for optimal patient care in chromosomal microarray testing.

PubMed

Wain, Karen E; Riggs, Erin; Hanson, Karen; Savage, Melissa; Riethmaier, Darlene; Muirhead, Andrea; Mitchell, Elyse; Packard, Bethanny Smith; Faucett, W Andrew

2012-10-01

The International Standards for Cytogenomic Arrays (ISCA) Consortium is a worldwide collaborative effort dedicated to optimizing patient care by improving the quality of chromosomal microarray testing. The primary effort of the ISCA Consortium has been the development of a database of copy number variants (CNVs) identified during the course of clinical microarray testing. This database is a powerful resource for clinicians, laboratories, and researchers, and can be utilized for a variety of applications, such as facilitating standardized interpretations of certain CNVs across laboratories or providing phenotypic information for counseling purposes when published data is sparse. A recognized limitation to the clinical utility of this database, however, is the quality of clinical information available for each patient. Clinical genetic counselors are uniquely suited to facilitate the communication of this information to the laboratory by virtue of their existing clinical responsibilities, case management skills, and appreciation of the evolving nature of scientific knowledge. We intend to highlight the critical role that genetic counselors play in ensuring optimal patient care through contributing to the clinical utility of the ISCA Consortium's database, as well as the quality of individual patient microarray reports provided by contributing laboratories. Current tools, paper and electronic forms, created to maximize this collaboration are shared. In addition to making a professional commitment to providing complete clinical information, genetic counselors are invited to become ISCA members and to become involved in the discussions and initiatives within the Consortium.

LS Bound based gene selection for DNA microarray data.

PubMed

Zhou, Xin; Mao, K Z

2005-04-15

One problem with discriminant analysis of DNA microarray data is that each sample is represented by quite a large number of genes, and many of them are irrelevant, insignificant or redundant to the discriminant problem at hand. Methods for selecting important genes are, therefore, of much significance in microarray data analysis. In the present study, a new criterion, called LS Bound measure, is proposed to address the gene selection problem. The LS Bound measure is derived from leave-one-out procedure of LS-SVMs (least squares support vector machines), and as the upper bound for leave-one-out classification results it reflects to some extent the generalization performance of gene subsets. We applied this LS Bound measure for gene selection on two benchmark microarray datasets: colon cancer and leukemia. We also compared the LS Bound measure with other evaluation criteria, including the well-known Fisher's ratio and Mahalanobis class separability measure, and other published gene selection algorithms, including Weighting factor and SVM Recursive Feature Elimination. The strength of the LS Bound measure is that it provides gene subsets leading to more accurate classification results than the filter method while its computational complexity is at the level of the filter method. A companion website can be accessed at http://www.ntu.edu.sg/home5/pg02776030/lsbound/. The website contains: (1) the source code of the gene selection algorithm; (2) the complete set of tables and figures regarding the experimental study; (3) proof of the inequality (9). ekzmao@ntu.edu.sg.

Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

PubMed

Carlson, Ruth I; Cattet, Marc R L; Sarauer, Bryan L; Nielsen, Scott E; Boulanger, John; Stenhouse, Gordon B; Janz, David M

2016-01-01

A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.

Efficacy of a novel PCR- and microarray-based method in diagnosis of a prosthetic joint infection

PubMed Central

2014-01-01

Background and purpose Polymerase chain reaction (PCR) methods enable detection and species identification of many pathogens. We assessed the efficacy of a new PCR and microarray-based platform for detection of bacteria in prosthetic joint infections (PJIs). Methods This prospective study involved 61 suspected PJIs in hip and knee prostheses and 20 negative controls. 142 samples were analyzed by Prove-it Bone and Joint assay. The laboratory staff conducting the Prove-it analysis were not aware of the results of microbiological culture and clinical findings. The results of the analysis were compared with diagnosis of PJIs defined according to the Musculoskeletal Infection Society (MSIS) criteria and with the results of microbiological culture. Results 38 of 61 suspected PJIs met the definition of PJI according to the MSIS criteria. Of the 38 patients, the PCR detected bacteria in 31 whereas bacterial culture was positive in 28 patients. 15 of the PJI patients were undergoing antimicrobial treatment as the samples for analysis were obtained. When antimicrobial treatment had lasted 4 days or more, PCR detected bacteria in 6 of the 9 patients, but positive cultures were noted in only 2 of the 9 patients. All PCR results for the controls were negative. Of the 61 suspected PJIs, there were false-positive PCR results in 6 cases. Interpretation The Prove-it assay was helpful in PJI diagnostics during ongoing antimicrobial treatment. Without preceding treatment with antimicrobials, PCR and microarray-based assay did not appear to give any additional information over culture. PMID:24564748

High-density fiber-optic DNA random microsphere array.

PubMed

Ferguson, J A; Steemers, F J; Walt, D R

2000-11-15

A high-density fiber-optic DNA microarray sensor was developed to monitor multiple DNA sequences in parallel. Microarrays were prepared by randomly distributing DNA probe-functionalized 3.1-microm-diameter microspheres in an array of wells etched in a 500-microm-diameter optical imaging fiber. Registration of the microspheres was performed using an optical encoding scheme and a custom-built imaging system. Hybridization was visualized using fluorescent-labeled DNA targets with a detection limit of 10 fM. Hybridization times of seconds are required for nanomolar target concentrations, and analysis is performed in minutes.

Single-Nucleotide Polymorphism-Microarray Ploidy Analysis of Paraffin-Embedded Products of Conception in Recurrent Pregnancy Loss Evaluations.

PubMed

Maslow, Bat-Sheva L; Budinetz, Tara; Sueldo, Carolina; Anspach, Erica; Engmann, Lawrence; Benadiva, Claudio; Nulsen, John C

2015-07-01

To compare the analysis of chromosome number from paraffin-embedded products of conception using single-nucleotide polymorphism (SNP) microarray with the recommended screening for the evaluation of couples presenting with recurrent pregnancy loss who do not have previous fetal cytogenetic data. We performed a retrospective cohort study including all women who presented for a new evaluation of recurrent pregnancy loss over a 2-year period (January 1, 2012, to December 31, 2013). All participants had at least two documented first-trimester losses and both the recommended screening tests and SNP microarray performed on at least one paraffin-embedded products of conception sample. Single-nucleotide polymorphism microarray identifies all 24 chromosomes (22 autosomes, X, and Y). Forty-two women with a total of 178 losses were included in the study. Paraffin-embedded products of conception from 62 losses were sent for SNP microarray. Single-nucleotide polymorphism microarray successfully diagnosed fetal chromosome number in 71% (44/62) of samples, of which 43% (19/44) were euploid and 57% (25/44) were noneuploid. Seven of 42 (17%) participants had abnormalities on recurrent pregnancy loss screening. The per-person detection rate for a cause of pregnancy loss was significantly higher in the SNP microarray (0.50; 95% confidence interval [CI] 0.36-0.64) compared with recurrent pregnancy loss evaluation (0.17; 95% CI 0.08-0.31) (P=.002). Participants with one or more euploid loss identified on paraffin-embedded products of conception were significantly more likely to have an abnormality on recurrent pregnancy loss screening than those with only noneuploid results (P=.028). The significance remained when controlling for age, number of losses, number of samples, and total pregnancies. These results suggest that SNP microarray testing of paraffin-embedded products of conception is a valuable tool for the evaluation of recurrent pregnancy loss in patients without prior fetal cytogenetic results. Recommended recurrent pregnancy loss screening was unnecessary in almost half the patients in our study. II.

Cell cycle arrest and gene expression profiling of testis in mice exposed to fluoride.

PubMed

Su, Kai; Sun, Zilong; Niu, Ruiyan; Lei, Ying; Cheng, Jing; Wang, Jundong

2017-05-01

Exposure to fluoride results in low reproductive capacity; however, the mechanism underlying the impact of fluoride on male productive system still remains obscure. To assess the potential toxicity in testis of mice administrated with fluoride, global genome microarray and real-time PCR were performed to detect and identify the altered transcriptions. The results revealed that 763 differentially expressed genes were identified, including 330 up-regulated and 433 down-regulated genes, which were involved in spermatogenesis, apoptosis, DNA damage, DNA replication, and cell differentiation. Twelve differential expressed genes were selected to confirm the microarray results using real-time PCR, and the result kept the same tendency with that of microarray. Furthermore, compared with the control group, more apoptotic spermatogenic cells were observed in the fluoride group, and the spermatogonium were markedly increased in S phase and decreased in G2/M phase by fluoride. Our findings suggested global genome microarray provides an insight into the reproductive toxicity induced by fluoride, and several important biological clues for further investigations. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1558-1565, 2017. © 2016 Wiley Periodicals, Inc.

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

PubMed Central

Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

2014-01-01

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

Spotting and validation of a genome wide oligonucleotide chip with duplicate measurement of each gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomassen, Mads; Skov, Vibe; Eiriksdottir, Freyja

2006-06-16

The quality of DNA microarray based gene expression data relies on the reproducibility of several steps in a microarray experiment. We have developed a spotted genome wide microarray chip with oligonucleotides printed in duplicate in order to minimise undesirable biases, thereby optimising detection of true differential expression. The validation study design consisted of an assessment of the microarray chip performance using the MessageAmp and FairPlay labelling kits. Intraclass correlation coefficient (ICC) was used to demonstrate that MessageAmp was significantly more reproducible than FairPlay. Further examinations with MessageAmp revealed the applicability of the system. The linear range of the chips wasmore » three orders of magnitude, the precision was high, as 95% of measurements deviated less than 1.24-fold from the expected value, and the coefficient of variation for relative expression was 13.6%. Relative quantitation was more reproducible than absolute quantitation and substantial reduction of variance was attained with duplicate spotting. An analysis of variance (ANOVA) demonstrated no significant day-to-day variation.« less

MASQOT: a method for cDNA microarray spot quality control

PubMed Central

Bylesjö, Max; Eriksson, Daniel; Sjödin, Andreas; Sjöström, Michael; Jansson, Stefan; Antti, Henrik; Trygg, Johan

2005-01-01

Background cDNA microarray technology has emerged as a major player in the parallel detection of biomolecules, but still suffers from fundamental technical problems. Identifying and removing unreliable data is crucial to prevent the risk of receiving illusive analysis results. Visual assessment of spot quality is still a common procedure, despite the time-consuming work of manually inspecting spots in the range of hundreds of thousands or more. Results A novel methodology for cDNA microarray spot quality control is outlined. Multivariate discriminant analysis was used to assess spot quality based on existing and novel descriptors. The presented methodology displays high reproducibility and was found superior in identifying unreliable data compared to other evaluated methodologies. Conclusion The proposed methodology for cDNA microarray spot quality control generates non-discrete values of spot quality which can be utilized as weights in subsequent analysis procedures as well as to discard spots of undesired quality using the suggested threshold values. The MASQOT approach provides a consistent assessment of spot quality and can be considered an alternative to the labor-intensive manual quality assessment process. PMID:16223442

A DNA microarray for identification of selected Korean birds based on mitochondrial cytochrome c oxidase I gene sequences.

PubMed

Chung, In-Hyuk; Yoo, Hye Sook; Eah, Jae-Yong; Yoon, Hyun-Kyu; Jung, Jin-Wook; Hwang, Seung Yong; Kim, Chang-Bae

2010-10-01

DNA barcoding with the gene encoding cytochrome c oxidase I (COI) in the mitochondrial genome has been proposed as a standard marker to identify and discover animal species. Some migratory wild birds are suspected of transmitting avian influenza and pose a threat to aircraft safety because of bird strikes. We have previously reported the COI gene sequences of 92 Korean bird species. In the present study, we developed a DNA microarray to identify 17 selected bird species on the basis of nucleotide diversity. We designed and synthesized 19 specific oligonucleotide probes; these probes were arrayed on a silylated glass slide. The length of the probes was 19-24 bps. The COI sequences amplified from the tissues of the selected birds were labeled with a fluorescent probe for microarray hybridization, and unique hybridization patterns were detected for each selected species. These patterns may be considered diagnostic patterns for species identification. This microarray system will provide a sensitive and a high-throughput method for identification of Korean birds.

[Expression of cell adhesion molecules in acute leukemia cell].

PubMed

Ju, Xiaoping; Peng, Min; Xu, Xiaoping; Lu, Shuqing; Li, Yao; Ying, Kang; Xie, Yi; Mao, Yumin; Xia, Fang

2002-11-01

To investigate the role of cell adhesion molecule in the development and extramedullary infiltration (EI) of acute leukemia. The expressions of neural cell adhesion molecule (NCAM) gene, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) genes in 25 acute leukemia patients bone marrow cells were detected by microarray and reverse transcriptase-polymerase chain reaction (RT-PCR). The expressions of NCAM, ICAM-1 and VCAM-1 gene were significantly higher in acute leukemia cells and leukemia cells with EI than in normal tissues and leukemia cells without EI, respectively, both by cDNA microarray and by RT-PCR. The cDNA microarray is a powerful technique in analysis of acute leukemia cells associated genes. High expressions of cell adhesion molecule genes might be correlated with leukemia pathogenesis and infiltration of acute leukemia cell.

Single cell transcriptomics of neighboring hyphae of Aspergillus niger

PubMed Central

2011-01-01

Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories. PMID:21816052

DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets.

PubMed

Husale, Sudhir; Persson, Henrik H J; Sahin, Ozgur

2009-12-24

Techniques to detect and quantify DNA and RNA molecules in biological samples have had a central role in genomics research. Over the past decade, several techniques have been developed to improve detection performance and reduce the cost of genetic analysis. In particular, significant advances in label-free methods have been reported. Yet detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. Here we report a unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label. Nanomechanical measurements on a microarray surface have sufficient background signal rejection to allow direct detection and counting of hybridized molecules. The digital response of the sensor provides a large dynamic range that is critical for gene expression profiling. We have measured differential expressions of microRNAs in tumour samples; such measurements have been shown to help discriminate between the tissue origins of metastatic tumours. Two hundred picograms of total RNA is found to be sufficient for this analysis. In addition, the limit of detection in pure samples is found to be one attomolar. These results suggest that nanomechanical read-out of microarrays promises attomolar-level sensitivity and large dynamic range for the analysis of gene expression, while eliminating biochemical manipulations, amplification and labelling.

Detection systems for carbapenemase gene identification should include the SME serine carbapenemase.

PubMed

Bush, Karen; Pannell, Megan; Lock, John L; Queenan, Anne Marie; Jorgensen, James H; Lee, Ryan M; Lewis, James S; Jarrett, Deidre

2013-01-01

Carbapenemase detection has become a major problem in hospitals that encounter outbreaks of infections caused by carbapenem-resistant Gram-negative bacteria. Rapid detection systems have been reported using multiplex PCR analyses and DNA microarray assays. Major carbapenemases that are detected by these systems include the KPC and OXA serine carbapenemases, and the IMP, VIM and NDM families of metallo-β-lactamases. However, increasing numbers of the SME serine carbapenemase are being reported from Serratia marcescens, especially from North and South America. These organisms differ from many of the other carbapenemase-producing pathogens in that they are generally susceptible to the expanded-spectrum cephalosporins ceftazidime and cefepime while retaining resistance to almost all other β-lactam antibiotics. Thus, multiplex PCR assays or DNA microarray testing of carbapenem-resistant S. marcescens isolates should include analyses for production of the SME carbapenemase. Confirmation of the presence of this enzyme may provide reassurance that oxyimino-cephalosporins can be considered for treatment of infections caused by these carbapenem-resistant pathogens. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Microbial Profiling of Combat Wound Infection through Detection Microarray and Next-Generation Sequencing

PubMed Central

Allen, Jonathan E.; Brown, Trevor S.; Gardner, Shea N.; McLoughlin, Kevin S.; Forsberg, Jonathan A.; Kirkup, Benjamin C.; Chromy, Brett A.; Luciw, Paul A.; Elster, Eric A.

2014-01-01

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden. PMID:24829242

Detection of high-risk mucosal human papillomavirus DNA in human specimens by a novel and sensitive multiplex PCR method combined with DNA microarray.

PubMed

Gheit, Tarik; Tommasino, Massimo

2011-01-01

Epidemiological and functional studies have clearly demonstrated that certain types of human papillomavirus (HPV) from the genus alpha of the HPV phylogenetic tree, referred to as high-risk (HR) types, are the etiological cause of cervical cancer. Several methods for HPV detection and typing have been developed, and their importance in clinical and epidemiological studies has been well demonstrated. However, comparative studies have shown that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. In this chapter, we describe a novel one-shot method for the detection and typing of 19 mucosal HR HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). The assay combines the advantages of the multiplex PCR methods, i.e., high sensitivity and the possibility to perform multiple amplifications in a single reaction, with an array primer extension (APEX) assay. The latter method offers the benefits of Sanger dideoxy sequencing with the high-throughput potential of the microarray. Initial studies have revealed that the assay is very sensitive in detecting multiple HPV infections.

[Microarray CGH: principle and use for constitutional disorders].

PubMed

Sanlaville, D; Lapierre, J M; Coquin, A; Turleau, C; Vermeesch, J; Colleaux, L; Borck, G; Vekemans, M; Aurias, A; Romana, S P

2005-10-01

Chips technology has allowed to miniaturize process making possible to realize in one step and using the same device a lot of chemical reactions. The application of this technology to molecular cytogenetics resulted in the development of comparative genomic hybridization (CGH) on microarrays technique. Using this technique it is possible to detect very small genetic imbalances anywhere in the genome. Its usefulness has been well documented in cancer and more recently in constitutional disorders. In particular it has been used to detect interstitial and subtelomeric submicroscopic imbalances, to characterize their size at the molecular level or to define the breakpoints of translocation. The challenge today is to transfer this technology in laboratory medicine. Nevertheless this technology remains expensive and the existence of numerous sequence polymorphisms makes its interpretation difficult. Finally its is unlikely that it will make karyotyping obsolete as it does not allow to detect balanced rearrangements which after meiotic segregation might result in genome imbalance in the progeny.

Cytokine-related genes and oxidation-related genes detected in preeclamptic placentas.

PubMed

Lee, Gui Se Ra; Joe, Yoon Seong; Kim, Sa Jin; Shin, Jong Chul

2010-10-01

To investigate cytokine- and oxidation-related genes for preeclampsia using DNA microarray analysis. Placentas were collected from 13 normal pregnancies and 13 patients with preeclampsia. Gene expression was studied using DNA microarray. Among significantly expressed genes, we focused on genes associated with cytokines and oxidation, and the results were confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). 415 genes out of 30,940 genes were altered by > or =2-fold in the microarray analysis. 121 up-regulated genes and 294 down-regulated genes were found to be in preeclamptic placenta. Six cytokine-related genes and 5 oxidation-related genes were found from among the 121 up-regulated genes. The cytokine-related genes studied included oncostatin M (OSM), fms-related tyrosine kinase (FLT1) and vascular endothelial growth factor A (VEGFA), and the oxidation-related genes studied included spermine oxidase (SMOX), l cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1), acetate dehydrogenase A (LDHA). These six genes were also significantly higher in placentas from patients with preeclampsia than in those from women with normal pregnancies. The placental tissue of patients with preeclampsia showed significantly higher mRNA expression of these six genes than the normal group, using QRT-PCR. DNA microarray analysis is one of the great methods for simultaneously detecting the functionally associated genes of preeclampsia. The cytokine-related genes such as OSM, FLT1 and VEGFA, and the oxidation-related genes such as LDHA, CYP26A1 and SMOX might prove to be the starting point in the elucidation of the pathogenesis of preeclampsia.

Application of Immunosignatures for Diagnosis of Valley Fever

PubMed Central

Navalkar, Krupa Arun; Johnston, Stephen Albert; Woodbury, Neal; Galgiani, John N.; Magee, D. Mitchell; Chicacz, Zbigniew

2014-01-01

Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives. An immunosignature is the pattern of antibody binding to random-sequence peptides on a peptide microarray. A 10,000-peptide microarray was first used to determine whether valley fever patients can be distinguished from 3 other cohorts with similar infections. After determining the VF-specific peptides, a small 96-peptide diagnostic array was created and tested. The performances of the 10,000-peptide array and the 96-peptide diagnostic array were compared to that of the ID diagnostic standard. The 10,000-peptide microarray classified the VF samples from the other 3 infections with 98% accuracy. It also classified VF false-negative patients with 100% sensitivity in a blinded test set versus 28% sensitivity for ID. The immunosignature microarray has potential for simultaneously distinguishing valley fever patients from those with other fungal or bacterial infections. The same 10,000-peptide array can diagnose VF false-negative patients with 100% sensitivity. The smaller 96-peptide diagnostic array was less specific for diagnosing false negatives. We conclude that the performance of the immunosignature diagnostic exceeds that of the existing standard, and the immunosignature can distinguish related infections and might be used in lieu of existing diagnostics. PMID:24964807

An Interview with Matthew P. Greving, PhD. Interview by Vicki Glaser.

PubMed

Greving, Matthew P

2011-10-01

Matthew P. Greving is Chief Scientific Officer at Nextval Inc., a company founded in early 2010 that has developed a discovery platform called MassInsight™.. He received his PhD in Biochemistry from Arizona State University, and prior to that he spent nearly 7 years working as a software engineer. This experience in solving complex computational problems fueled his interest in developing technologies and algorithms related to acquisition and analysis of high-dimensional biochemical data. To address the existing problems associated with label-based microarray readouts, he beganwork on a technique for label-free mass spectrometry (MS) microarray readout compatible with both matrix-assisted laser/desorption ionization (MALDI) and matrix-free nanostructure initiator mass spectrometry (NIMS). This is the core of Nextval’s MassInsight technology, which utilizes picoliter noncontact deposition of high-density arrays on mass-readout substrates along with computational algorithms for high-dimensional data processingand reduction.

Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

PubMed Central

Deutsch, Eric W; Ball, Catherine A; Berman, Jules J; Bova, G Steven; Brazma, Alvis; Bumgarner, Roger E; Campbell, David; Causton, Helen C; Christiansen, Jeffrey H; Daian, Fabrice; Dauga, Delphine; Davidson, Duncan R; Gimenez, Gregory; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Herrmann, Bernhard G; Johnson, Michael H; Korb, Martin; Mills, Jason C; Oudes, Asa J; Parkinson, Helen E; Pascal, Laura E; Pollet, Nicolas; Quackenbush, John; Ramialison, Mirana; Ringwald, Martin; Salgado, David; Sansone, Susanna-Assunta; Sherlock, Gavin; Stoeckert, Christian J; Swedlow, Jason; Taylor, Ronald C; Walashek, Laura; Warford, Anthony; Wilkinson, David G; Zhou, Yi; Zon, Leonard I; Liu, Alvin Y; True, Lawrence D

2015-01-01

One purpose of the biomedical literature is to report results in sufficient detail so that the methods of data collection and analysis can be independently replicated and verified. Here we present for consideration a minimum information specification for gene expression localization experiments, called the “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. Data specifications like MIAME and MISFISHIE specify the information content without dictating a format for encoding that information. The MISFISHIE specification describes six types of information that should be provided for each experiment: Experimental Design, Biomaterials and Treatments, Reporters, Staining, Imaging Data, and Image Characterizations. This specification has benefited the consortium within which it was initially developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal. PMID:18327244

CoPub: a literature-based keyword enrichment tool for microarray data analysis.

PubMed

Frijters, Raoul; Heupers, Bart; van Beek, Pieter; Bouwhuis, Maurice; van Schaik, René; de Vlieg, Jacob; Polman, Jan; Alkema, Wynand

2008-07-01

Medline is a rich information source, from which links between genes and keywords describing biological processes, pathways, drugs, pathologies and diseases can be extracted. We developed a publicly available tool called CoPub that uses the information in the Medline database for the biological interpretation of microarray data. CoPub allows batch input of multiple human, mouse or rat genes and produces lists of keywords from several biomedical thesauri that are significantly correlated with the set of input genes. These lists link to Medline abstracts in which the co-occurring input genes and correlated keywords are highlighted. Furthermore, CoPub can graphically visualize differentially expressed genes and over-represented keywords in a network, providing detailed insight in the relationships between genes and keywords, and revealing the most influential genes as highly connected hubs. CoPub is freely accessible at http://services.nbic.nl/cgi-bin/copub/CoPub.pl.

MSL: A Measure to Evaluate Three-dimensional Patterns in Gene Expression Data

PubMed Central

Gutiérrez-Avilés, David; Rubio-Escudero, Cristina

2015-01-01

Microarray technology is highly used in biological research environments due to its ability to monitor the RNA concentration levels. The analysis of the data generated represents a computational challenge due to the characteristics of these data. Clustering techniques are widely applied to create groups of genes that exhibit a similar behavior. Biclustering relaxes the constraints for grouping, allowing genes to be evaluated only under a subset of the conditions. Triclustering appears for the analysis of longitudinal experiments in which the genes are evaluated under certain conditions at several time points. These triclusters provide hidden information in the form of behavior patterns from temporal experiments with microarrays relating subsets of genes, experimental conditions, and time points. We present an evaluation measure for triclusters called Multi Slope Measure, based on the similarity among the angles of the slopes formed by each profile formed by the genes, conditions, and times of the tricluster. PMID:26124630

Exome sequencing of a multigenerational human pedigree.

PubMed

Hedges, Dale J; Hedges, Dale; Burges, Dan; Powell, Eric; Almonte, Cherylyn; Huang, Jia; Young, Stuart; Boese, Benjamin; Schmidt, Mike; Pericak-Vance, Margaret A; Martin, Eden; Zhang, Xinmin; Harkins, Timothy T; Züchner, Stephan

2009-12-14

Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.

A preliminary result of three-dimensional microarray technology to gene analysis with endoscopic ultrasound-guided fine-needle aspiration specimens and pancreatic juices

PubMed Central

2010-01-01

Background Analysis of gene expression and gene mutation may add information to be different from ordinary pathological tissue diagnosis. Since samples obtained endoscopically are very small, it is desired that more sensitive technology is developed for gene analysis. We investigated whether gene expression and gene mutation analysis by newly developed ultra-sensitive three-dimensional (3D) microarray is possible using small amount samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens and pancreatic juices. Methods Small amount samples from 17 EUS-FNA specimens and 16 pancreatic juices were obtained. After nucleic acid extraction, the samples were amplified with labeling and analyzed by the 3D microarray. Results The analyzable rate with the microarray was 46% (6/13) in EUS-FNA specimens of RNAlater® storage, and RNA degradations were observed in all the samples of frozen storage. In pancreatic juices, the analyzable rate was 67% (4/6) in frozen storage samples and 20% (2/10) in RNAlater® storage. EUS-FNA specimens were classified into cancer and non-cancer by gene expression analysis and K-ras codon 12 mutations were also detected using the 3D microarray. Conclusions Gene analysis from small amount samples obtained endoscopically was possible by newly developed 3D microarray technology. High quality RNA from EUS-FNA samples were obtained and remained in good condition only using RNA stabilizer. In contrast, high quality RNA from pancreatic juice samples were obtained only in frozen storage without RNA stabilizer. PMID:20416107

Robust gene selection methods using weighting schemes for microarray data analysis.

PubMed

Kang, Suyeon; Song, Jongwoo

2017-09-02

A common task in microarray data analysis is to identify informative genes that are differentially expressed between two different states. Owing to the high-dimensional nature of microarray data, identification of significant genes has been essential in analyzing the data. However, the performances of many gene selection techniques are highly dependent on the experimental conditions, such as the presence of measurement error or a limited number of sample replicates. We have proposed new filter-based gene selection techniques, by applying a simple modification to significance analysis of microarrays (SAM). To prove the effectiveness of the proposed method, we considered a series of synthetic datasets with different noise levels and sample sizes along with two real datasets. The following findings were made. First, our proposed methods outperform conventional methods for all simulation set-ups. In particular, our methods are much better when the given data are noisy and sample size is small. They showed relatively robust performance regardless of noise level and sample size, whereas the performance of SAM became significantly worse as the noise level became high or sample size decreased. When sufficient sample replicates were available, SAM and our methods showed similar performance. Finally, our proposed methods are competitive with traditional methods in classification tasks for microarrays. The results of simulation study and real data analysis have demonstrated that our proposed methods are effective for detecting significant genes and classification tasks, especially when the given data are noisy or have few sample replicates. By employing weighting schemes, we can obtain robust and reliable results for microarray data analysis.

Recommendations for the use of microarrays in prenatal diagnosis.

PubMed

Suela, Javier; López-Expósito, Isabel; Querejeta, María Eugenia; Martorell, Rosa; Cuatrecasas, Esther; Armengol, Lluis; Antolín, Eugenia; Domínguez Garrido, Elena; Trujillo-Tiebas, María José; Rosell, Jordi; García Planells, Javier; Cigudosa, Juan Cruz

2017-04-07

Microarray technology, recently implemented in international prenatal diagnosis systems, has become one of the main techniques in this field in terms of detection rate and objectivity of the results. This guideline attempts to provide background information on this technology, including technical and diagnostic aspects to be considered. Specifically, this guideline defines: the different prenatal sample types to be used, as well as their characteristics (chorionic villi samples, amniotic fluid, fetal cord blood or miscarriage tissue material); variant reporting policies (including variants of uncertain significance) to be considered in informed consents and prenatal microarray reports; microarray limitations inherent to the technique and which must be taken into account when recommending microarray testing for diagnosis; a detailed clinical algorithm recommending the use of microarray testing and its introduction into routine clinical practice within the context of other genetic tests, including pregnancies in families with a genetic history or specific syndrome suspicion, first trimester increased nuchal translucency or second trimester heart malformation and ultrasound findings not related to a known or specific syndrome. This guideline has been coordinated by the Spanish Association for Prenatal Diagnosis (AEDP, «Asociación Española de Diagnóstico Prenatal»), the Spanish Human Genetics Association (AEGH, «Asociación Española de Genética Humana») and the Spanish Society of Clinical Genetics and Dysmorphology (SEGCyD, «Sociedad Española de Genética Clínica y Dismorfología»). Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

NASA Astrophysics Data System (ADS)

Shepard, Jason R. E.

2009-05-01

The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

Detection of microbial communities in continuous and discontinuous membrane bioreactor using high-density oligonucleotide Microarray

NASA Astrophysics Data System (ADS)

Duan, Liang; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W.

2010-11-01

This study compared the whole composition of microbial communities in continuous-flow (MBR) and batch-fed (discontinuous) (MSBR) aerobic membrane bioreactors using high-density universal 16S rRNA Microarray. The array includes 506,944 probes targeted to 8935 clusters in 16S rRNA gene sequences. The Microarray results showed that both MBR and MSBR had high microbial diversity. 1126 and 1002 bacterial subfamilies were detected and can separate as 37 and 32 phyla in MBR and MSBR, respectively. Proteobacteria was the predominant phylum, 703 and 597 subfamilies were found in two systems, which constituted 62.4% and 59.6% of the whole bacteria. Gamma- and Alpha-were the dominant classes in Proteobacteria. It occupied 38.1% and 26.3%, 31.2% and 39.2% for MBR and MSBR, respectively. Bacteroidetes, Firmicutes and Actinobacteria were the subdominant groups, occupying around 9.4% and 7.6%, 6.1% and 6.5%, 6.0% and 9.0% of the total bacteria in two reactors. Some bacterial groups such as Acidobacteria, Chloroflexi, Cyanobacteria, Verrucomicrobia and Spirochaetes also found more than 15 subfamilies. All the results indicated that the MBR system had more bacteria community diversity than MSBR's. Moreover, it was very interested that MBR and MSBR had almost the same bacterial composition except Enterobacteriaceae. 63 OTUs of Enterobacteriaceae were detected in MBR, while just 10 OTUs were found in MSBR. That's one of the reasons leading to the difference of the bacterial diversity between two bioreactors.

Implementation of spectral clustering on microarray data of carcinoma using k-means algorithm

NASA Astrophysics Data System (ADS)

Frisca, Bustamam, Alhadi; Siswantining, Titin

2017-03-01

Clustering is one of data analysis methods that aims to classify data which have similar characteristics in the same group. Spectral clustering is one of the most popular modern clustering algorithms. As an effective clustering technique, spectral clustering method emerged from the concepts of spectral graph theory. Spectral clustering method needs partitioning algorithm. There are some partitioning methods including PAM, SOM, Fuzzy c-means, and k-means. Based on the research that has been done by Capital and Choudhury in 2013, when using Euclidian distance k-means algorithm provide better accuracy than PAM algorithm. So in this paper we use k-means as our partition algorithm. The major advantage of spectral clustering is in reducing data dimension, especially in this case to reduce the dimension of large microarray dataset. Microarray data is a small-sized chip made of a glass plate containing thousands and even tens of thousands kinds of genes in the DNA fragments derived from doubling cDNA. Application of microarray data is widely used to detect cancer, for the example is carcinoma, in which cancer cells express the abnormalities in his genes. The purpose of this research is to classify the data that have high similarity in the same group and the data that have low similarity in the others. In this research, Carcinoma microarray data using 7457 genes. The result of partitioning using k-means algorithm is two clusters.

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Plasmonically amplified fluorescence bioassay with microarray format

NASA Astrophysics Data System (ADS)

Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.

2015-05-01

Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

The role of metalloendopeptidases in oropharyngeal carcinomas assessed by tissue microarray.

PubMed

Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Noguti, Juliana; Oshima, Celina T F; Ihara, Silvia S M; Franco, Marcello F

2011-01-01

The goal of this study was to investigate the expression of some metalloendopeptidases in squamous cell carcinomas of the oropharynx as well as its relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of EP24.15 and EP24.16 by means of tissue microarrays. Expression of EP24.15 or EP24.16 was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinomas of the oropharynx. In summary, our results support the notion that EP24.15 and EP24.16 are expressed in carcinoma of the oropharynx; however, these do not appear to be suitable biomarkers for histological grading, disease stage or recurrence as depicted by tissue microarrays and immunohistochemistry.

A Discovery Resource of Rare Copy Number Variations in Individuals with Autism Spectrum Disorder

PubMed Central

Prasad, Aparna; Merico, Daniele; Thiruvahindrapuram, Bhooma; Wei, John; Lionel, Anath C.; Sato, Daisuke; Rickaby, Jessica; Lu, Chao; Szatmari, Peter; Roberts, Wendy; Fernandez, Bridget A.; Marshall, Christian R.; Hatchwell, Eli; Eis, Peggy S.; Scherer, Stephen W.

2012-01-01

The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection. PMID:23275889

Application of the Gini correlation coefficient to infer regulatory relationships in transcriptome analysis.

PubMed

Ma, Chuang; Wang, Xiangfeng

2012-09-01

One of the computational challenges in plant systems biology is to accurately infer transcriptional regulation relationships based on correlation analyses of gene expression patterns. Despite several correlation methods that are applied in biology to analyze microarray data, concerns regarding the compatibility of these methods with the gene expression data profiled by high-throughput RNA transcriptome sequencing (RNA-Seq) technology have been raised. These concerns are mainly due to the fact that the distribution of read counts in RNA-Seq experiments is different from that of fluorescence intensities in microarray experiments. Therefore, a comprehensive evaluation of the existing correlation methods and, if necessary, introduction of novel methods into biology is appropriate. In this study, we compared four existing correlation methods used in microarray analysis and one novel method called the Gini correlation coefficient on previously published microarray-based and sequencing-based gene expression data in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). The comparisons were performed on more than 11,000 regulatory relationships in Arabidopsis, including 8,929 pairs of transcription factors and target genes. Our analyses pinpointed the strengths and weaknesses of each method and indicated that the Gini correlation can compensate for the shortcomings of the Pearson correlation, the Spearman correlation, the Kendall correlation, and the Tukey's biweight correlation. The Gini correlation method, with the other four evaluated methods in this study, was implemented as an R package named rsgcc that can be utilized as an alternative option for biologists to perform clustering analyses of gene expression patterns or transcriptional network analyses.

Application of the Gini Correlation Coefficient to Infer Regulatory Relationships in Transcriptome Analysis[W][OA

PubMed Central

Ma, Chuang; Wang, Xiangfeng

2012-01-01

One of the computational challenges in plant systems biology is to accurately infer transcriptional regulation relationships based on correlation analyses of gene expression patterns. Despite several correlation methods that are applied in biology to analyze microarray data, concerns regarding the compatibility of these methods with the gene expression data profiled by high-throughput RNA transcriptome sequencing (RNA-Seq) technology have been raised. These concerns are mainly due to the fact that the distribution of read counts in RNA-Seq experiments is different from that of fluorescence intensities in microarray experiments. Therefore, a comprehensive evaluation of the existing correlation methods and, if necessary, introduction of novel methods into biology is appropriate. In this study, we compared four existing correlation methods used in microarray analysis and one novel method called the Gini correlation coefficient on previously published microarray-based and sequencing-based gene expression data in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). The comparisons were performed on more than 11,000 regulatory relationships in Arabidopsis, including 8,929 pairs of transcription factors and target genes. Our analyses pinpointed the strengths and weaknesses of each method and indicated that the Gini correlation can compensate for the shortcomings of the Pearson correlation, the Spearman correlation, the Kendall correlation, and the Tukey’s biweight correlation. The Gini correlation method, with the other four evaluated methods in this study, was implemented as an R package named rsgcc that can be utilized as an alternative option for biologists to perform clustering analyses of gene expression patterns or transcriptional network analyses. PMID:22797655

Oligonucleotide microarray for subtyping of influenza A viruses

NASA Astrophysics Data System (ADS)

Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

2012-02-01

Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

Nonlinear matching measure for the analysis of on-off type DNA microarray images

NASA Astrophysics Data System (ADS)

Kim, Jong D.; Park, Misun; Kim, Jongwon

2003-07-01

In this paper, we propose a new nonlinear matching measure for automatic analysis of the on-off type DNA microarray images in which the hybridized spots are detected by the template matching method. The targeting spots of HPV DNA chips are designed for genotyping the human papilloma virus(HPV). The proposed measure is obtained by binarythresholding over the whole template region and taking the number of white pixels inside the spotted area. This measure is evaluated in terms of the accuracy of the estimated marker location to show better performance than the normalized covariance.

Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE).

PubMed

Yoo, Gu; Bong, Ji-Hong; Kim, Sinyoung; Jose, Joachim; Pyun, Jae-Chul

2014-07-15

A microarray-based immunoassay for the detection of autoantibodies against Ro protein was developed using Escherichia coli with autodisplayed Ro proteins (Ro(+)-E. coli). Patient serum usually contains various antibodies against the outer membrane components of E. coli as well as autoantibodies against the Ro protein. Therefore, the conventional immunoassay based on Ro(+)-E. coli requires both wild type E. coli (blank test) and Ro(+)-E. coli, and both strains of E. coli must be prepared in situ for each individual test serum. In this study, we tested the feasibility of using several types of animal sera as a replacement for individual human sera. An immunoassay without the blank test was developed using Ro(+)-E. coli by (1) blocking with rabbit serum, and (2) cleaving the Fc region from antibodies using papain. Modified E. coli with autodisplayed Ro protein was immobilized to a surface-modified microplate and the applicability of the immunoassay without the blank test was demonstrated using sera from patients with systemic lupus erythematosus (SLE). Using this approach, a microarray-based fluorescence immunoassay with immobilized Ro(+)-E. coli was able to detect anti-Ro autoantibodies in SLE patient sera with high specificity and selectivity and improved efficiency. Copyright © 2014 Elsevier B.V. All rights reserved.

Screening for Intellectual Disability Using High-Resolution CMA Technology in a Retrospective Cohort from Central Brazil

PubMed Central

Pereira, Rodrigo Roncato; Pinto, Irene Plaza; Minasi, Lysa Bernardes; de Melo, Aldaires Vieira; da Cruz e Cunha, Damiana Mirian; Cruz, Alex Silva; Ribeiro, Cristiano Luiz; da Silva, Cláudio Carlos; de Melo e Silva, Daniela; da Cruz, Aparecido Divino

2014-01-01

Intellectual disability is a complex, variable, and heterogeneous disorder, representing a disabling condition diagnosed worldwide, and the etiologies are multiple and highly heterogeneous. Microscopic chromosomal abnormalities and well-characterized genetic conditions are the most common causes of intellectual disability. Chromosomal Microarray Analysis analyses have made it possible to identify putatively pathogenic copy number variation that could explain the molecular etiology of intellectual disability. The aim of the current study was to identify possible submicroscopic genomic alterations using a high-density chromosomal microarray in a retrospective cohort of patients with otherwise undiagnosable intellectual disabilities referred by doctors from the public health system in Central Brazil. The CytoScan HD technology was used to detect changes in the genome copy number variation of patients who had intellectual disability and a normal karyotype. The analysis detected 18 CNVs in 60% of patients. Pathogenic CNVs represented about 22%, so it was possible to propose the etiology of intellectual disability for these patients. Likely pathogenic and unknown clinical significance CNVs represented 28% and 50%, respectively. Inherited and de novo CNVs were equally distributed. We report the nature of CNVs in patients from Central Brazil, representing a population not yet screened by microarray technologies. PMID:25061755

Shrinkage covariance matrix approach based on robust trimmed mean in gene sets detection

NASA Astrophysics Data System (ADS)

Karjanto, Suryaefiza; Ramli, Norazan Mohamed; Ghani, Nor Azura Md; Aripin, Rasimah; Yusop, Noorezatty Mohd

2015-02-01

Microarray involves of placing an orderly arrangement of thousands of gene sequences in a grid on a suitable surface. The technology has made a novelty discovery since its development and obtained an increasing attention among researchers. The widespread of microarray technology is largely due to its ability to perform simultaneous analysis of thousands of genes in a massively parallel manner in one experiment. Hence, it provides valuable knowledge on gene interaction and function. The microarray data set typically consists of tens of thousands of genes (variables) from just dozens of samples due to various constraints. Therefore, the sample covariance matrix in Hotelling's T2 statistic is not positive definite and become singular, thus it cannot be inverted. In this research, the Hotelling's T2 statistic is combined with a shrinkage approach as an alternative estimation to estimate the covariance matrix to detect significant gene sets. The use of shrinkage covariance matrix overcomes the singularity problem by converting an unbiased to an improved biased estimator of covariance matrix. Robust trimmed mean is integrated into the shrinkage matrix to reduce the influence of outliers and consequently increases its efficiency. The performance of the proposed method is measured using several simulation designs. The results are expected to outperform existing techniques in many tested conditions.

Diazonium-protein adducts for graphite electrode microarrays modification: direct and addressed electrochemical immobilization.

PubMed

Corgier, Benjamin P; Marquette, Christophe A; Blum, Loïc J

2005-12-28

Diazonium cation electrodeposition was investigated for the direct and electro-addressed immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified proteins. Screen-printed (SP) graphite electrode microarrays were studied as active support for this immobilization. A 10-microelectrode (eight working electrodes, 0.2 mm2 each; one reference; and one auxiliary) setup was used to study the addressing possibilities of the method. These electrode microarrays were shown to be able to covalently graft diazonium cations through electrochemical reduction. Cyclic voltammetry and X-ray photoelectron spectroscopy were used to characterize the electrochemical grafting onto our SP graphite surface and suggested that a diazonium monolayer was deposited. Rabbit and human immunoglobulins (IgGs) were then chemically coupled to an aniline derivative (4-carboxymethylaniline), followed by diazotation to form an aryl diazonium function available for the electrodeposition. These modified proteins were both successfully electro-addressed at the surface of the graphite electrodes without cross-talk or interference. The immuno-biochip obtained using this novel approach enabled the specific detection of anti-rabbit IgG antibodies with a detection limit of 50 fmol of protein. A promising strategy to immobilize markedly different biological entities was then presented, providing an excellent spatial specificity of the electro-addressing.

Hybrid feature selection algorithm using symmetrical uncertainty and a harmony search algorithm

NASA Astrophysics Data System (ADS)

Salameh Shreem, Salam; Abdullah, Salwani; Nazri, Mohd Zakree Ahmad

2016-04-01

Microarray technology can be used as an efficient diagnostic system to recognise diseases such as tumours or to discriminate between different types of cancers in normal tissues. This technology has received increasing attention from the bioinformatics community because of its potential in designing powerful decision-making tools for cancer diagnosis. However, the presence of thousands or tens of thousands of genes affects the predictive accuracy of this technology from the perspective of classification. Thus, a key issue in microarray data is identifying or selecting the smallest possible set of genes from the input data that can achieve good predictive accuracy for classification. In this work, we propose a two-stage selection algorithm for gene selection problems in microarray data-sets called the symmetrical uncertainty filter and harmony search algorithm wrapper (SU-HSA). Experimental results show that the SU-HSA is better than HSA in isolation for all data-sets in terms of the accuracy and achieves a lower number of genes on 6 out of 10 instances. Furthermore, the comparison with state-of-the-art methods shows that our proposed approach is able to obtain 5 (out of 10) new best results in terms of the number of selected genes and competitive results in terms of the classification accuracy.

Framework for Parallel Preprocessing of Microarray Data Using Hadoop

PubMed Central

2018-01-01

Nowadays, microarray technology has become one of the popular ways to study gene expression and diagnosis of disease. National Center for Biology Information (NCBI) hosts public databases containing large volumes of biological data required to be preprocessed, since they carry high levels of noise and bias. Robust Multiarray Average (RMA) is one of the standard and popular methods that is utilized to preprocess the data and remove the noises. Most of the preprocessing algorithms are time-consuming and not able to handle a large number of datasets with thousands of experiments. Parallel processing can be used to address the above-mentioned issues. Hadoop is a well-known and ideal distributed file system framework that provides a parallel environment to run the experiment. In this research, for the first time, the capability of Hadoop and statistical power of R have been leveraged to parallelize the available preprocessing algorithm called RMA to efficiently process microarray data. The experiment has been run on cluster containing 5 nodes, while each node has 16 cores and 16 GB memory. It compares efficiency and the performance of parallelized RMA using Hadoop with parallelized RMA using affyPara package as well as sequential RMA. The result shows the speed-up rate of the proposed approach outperforms the sequential approach and affyPara approach. PMID:29796018

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Galectins are human milk glycan receptors

PubMed Central

Noll, Alexander J; Gourdine, Jean-Philippe; Yu, Ying; Lasanajak, Yi; Smith, David F; Cummings, Richard D

2016-01-01

The biological recognition of human milk glycans (HMGs) is poorly understood. Because HMGs are rich in galactose we explored whether they might interact with human galectins, which bind galactose-containing glycans and are highly expressed in epithelial cells and other cell types. We screened a number of human galectins for their binding to HMGs on a shotgun glycan microarray consisting of 247 HMGs derived from human milk, as well as to a defined HMG microarray. Recombinant human galectins (hGal)-1, -3, -4, -7, -8 and -9 bound selectively to glycans, with each galectin recognizing a relatively unique binding motif; by contrast hGal-2 did not recognize HMGs, but did bind to the human blood group A Type 2 determinants on other microarrays. Unlike other galectins, hGal-7 preferentially bound to glycans expressing a terminal Type 1 (Galβ1-3GlcNAc) sequence, a motif that had eluded detection on non-HMG glycan microarrays. Interactions with HMGs were confirmed in a solution setting by isothermal titration microcalorimetry and hapten inhibition experiments. These results demonstrate that galectins selectively bind to HMGs and suggest the possibility that galectin–HMG interactions may play a role in infant immunity. PMID:26747425

Estimating differential expression from multiple indicators

PubMed Central

Ilmjärv, Sten; Hundahl, Christian Ansgar; Reimets, Riin; Niitsoo, Margus; Kolde, Raivo; Vilo, Jaak; Vasar, Eero; Luuk, Hendrik

2014-01-01

Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR. PMID:24586062

Development and application of a DNA microarray-based yeast two-hybrid system

PubMed Central

Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

2013-01-01

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays

PubMed Central

Fixe, F.; Dufva, M.; Telleman, P.; Christensen, C. B. V.

2004-01-01

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray™ slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques. PMID:14718554

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

Aberrant Expression of Calretinin, D2-40 and Mesothelin in Mucinous and Non-Mucinous Colorectal Carcinomas and Relation to Clinicopathological Features and Prognosis.

PubMed

Foda, Abd AlRahman Mohammad; El-Hawary, Amira Kamal; Hamed, Hazem

2016-10-01

CRC is a heterogeneous disease in terms of morphology, invasive behavior, metastatic capacity, and clinical outcome. Recently, many so-called mesothelial markers, including calretinin, D2-40, WT1, thrombomodulin, mesothelin, and others, have been certified. The aim of this study was to assess the immunohistochemical expression of calretinin and other mesothelial markers (D2-40 and mesothelin) in colorectal mucinous adenocarcinoma (MA) and non mucinous adenocarcinoma (NMA) specimens and relation to clinicopathological features and prognosis using manual tissue microarray technique. We studied tumor tissue specimens from 150 patients with colorectal MA and NMA who underwent radical surgery from January 2007 to January 2012. High-density manual tissue microarrays were constructed using a modified mechanical pencil tip technique, and paraffin sections were submitted for immunohistochemistry using Calretinin, D2-40 and mesothelin expressions. We found that NMA showed significantly more calretinin and D2-40 expression than MA In contrast, no statistically significant difference between NMA and MA was detected in mesothelin expression. There were no statistically significant relations between any of the clinicopathological or histological parameters and any of the three markers. In a univariate analysis, neither calretinin nor D2-40 expressions showed any significant relations to DFS or OS. However, mesothelin luminal expression was significantly associated with worse DFS. Multivariate Cox regression analysis proved that luminal mesothelin expression was an independent negative prognostic factor in NMA. In conclusion, Calretinin, D2-40 and mesothelin are aberrantly expressed in a proportion of CRC cases with more expression in NMA than MA. Aberrant expression of these mesothelial markers was not associated with clinicopathological or histological features of CRCs. Only mesothelin expression appears to be a strong predictor of adverse prognosis.

Soluble FLT-1 rules placental destiny.

PubMed

Yamashita, Michiko; Kumasawa, Keiichi; Nakamura, Hitomi; Kimura, Tadashi

2018-02-19

Placenta previa is an abnormality in which the placenta covers the internal uterine os, and it can cause serious morbidity and mortality in both mother and fetus due to catastrophic hemorrhage. Some pregnant women recover from placenta previa due to a phenomenon called "migration." However, the mechanism of "migration" of the placenta has not been elucidated. Human placentas were collected from patients with placenta previa and those with no abnormal placentation (control). A microarray analysis was performed to detect the genes up- or down-regulated only in the caudal part in the previa group. Specific mRNA expression was evaluated using real-time quantitative reverse transcription PCR (qRT-PCR). Unilateral uterine artery ablation of 8.5 dpc mice was performed to reproduce the reduction of placental blood supply, and weights of the placentas and fetuses were evaluated in 18.5 dpc. Specific mRNA expression was also evaluated in mice placentas. According to the result of the microarray analysis, we focused on soluble fms-like tyrosine kinase-1 (sFLT-1) and hypoxia-inducible factor-1 (HIF-1) alpha. The sFLT-1 expression level is locally high in the caudal part of the human placenta in patients with placenta previa. In mice experiments, the weights of the placentas and fetuses were significantly smaller in the ablation side than those in the control side, and the sFlt-1 expression level was significantly higher in the ablation side than in the control side. Our study suggests that "migration" of the placenta is derived from placental degeneration at the caudal part of the placenta, and sFlt-1 plays a role in this placental degeneration. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated in Poland.

PubMed

Januszkiewicz, Aleksandra; Rastawicki, Waldemar

2016-08-26

Shiga toxin-producing Escherichia coli (STEC) strains also called verotoxin-producing E. coli (VTEC) represent one of the most important groups of food-borne pathogens that can cause several human diseases such as hemorrhagic colitis (HC) and hemolytic - uremic syndrome (HUS) worldwide. The ability of STEC strains to cause disease is associated with the presence of wide range of identified and putative virulence factors including those encoding Shiga toxin. In this study, we examined the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. A total of 71 Shiga toxin-producing E. coli strains isolated from human, cattle and food over the years 1996-2010 were characterized by microarray and PCR detection of virulence genes. As stx1a subtype was present in all of the tested Shiga toxin 1 producing E. coli strains, a greater diversity of subtypes was found in the gene stx2, which occurred in five subtypes: stx2a, stx2b, stx2c, stx2d, stx2g. Among STEC O157 strains we observed conserved core set of 14 virulence factors, stable in bacteria genome at long intervals of time. There was one cattle STEC isolate which possessed verotoxin gene as well as sta1 gene encoded heat-stable enterotoxin STIa characteristic for enterotoxigenic E. coli. To the best of our knowledge, this is the first comprehensive analysis of virulence gene profiles identified in STEC strains isolated from human, cattle and food in Poland. The results obtained using microarrays technology confirmed high effectiveness of this method in determining STEC virulotypes which provides data suitable for molecular risk assessment of the potential virulence of this bacteria. virulence factors including those encoding Shiga toxin. In this study, we examined the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. A total of 71 Shiga toxin-producing E. coli strains isolated from human, cattle and food over the years 1996-2010 were characterized by microarray and PCR detection of virulence genes. As stx1a subtype was present in all of the tested Shiga toxin 1 producing E. coli strains, a greater diversity of subtypes was found in the gene stx2, which occurred in five subtypes: stx2a, stx2b, stx2c, stx2d, stx2g. Among STEC O157 strains we observed conserved core set of 14 virulence factors, stable in bacteria genome at long intervals of time. There was one cattle STEC isolate which possessed verotoxin gene as well as sta1 gene encoded heat-stable enterotoxin STIa characteristic for enterotoxigenic E. coli. To the best of our knowledge, this is the first comprehensive analysis of virulence gene profiles identified in STEC strains isolated from human, cattle and food in Poland. The results obtained using microarrays technology confirmed high effectiveness of this method in determining STEC virulotypes which provides data suitable for molecular risk assessment of the potential virulence of this bacteria.

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

PubMed Central

Ai, Lin; Chen, Jun-Hu; Chen, Shao-Hong; Zhang, Yong-Nian; Cai, Yu-Chun; Zhu, Xing-Quan; Zhou, Xiao-Nong

2012-01-01

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening. PMID:23209851

A 256 pixel magnetoresistive biosensor microarray in 0.18μm CMOS

PubMed Central

Hall, Drew A.; Gaster, Richard S.; Makinwa, Kofi; Wang, Shan X.; Murmann, Boris

2014-01-01

Magnetic nanotechnologies have shown significant potential in several areas of nanomedicine such as imaging, therapeutics, and early disease detection. Giant magnetoresistive spin-valve (GMR SV) sensors coupled with magnetic nanotags (MNTs) possess great promise as ultra-sensitive biosensors for diagnostics. We report an integrated sensor interface for an array of 256 GMR SV biosensors designed in 0.18 μm CMOS. Arranged like an imager, each of the 16 column level readout channels contains an analog front- end and a compact ΣΔ modulator (0.054 mm2) with 84 dB of dynamic range and an input referred noise of 49 nT/√Hz. Performance is demonstrated through detection of an ovarian cancer biomarker, secretory leukocyte peptidase inhibitor (SLPI), spiked at concentrations as low as 10 fM. This system is designed as a replacement for optical protein microarrays while also providing real-time kinetics monitoring. PMID:24761029

Breast Reference Set Application: Karen Anderson-ASU (2014) — EDRN Public Portal

Cancer.gov

In order to increase the predictive value of tumor-specific antibodies for use as immunodiagnostics, our EDRN BDL has developed a novel protein microarray technology, termed Nucleic Acid Protein Programmable Array (NAPPA), which circumvents many of the limitations of traditional protein microarrays. NAPPA arrays are generated by printing full-length cDNAs encoding the target proteins at each feature of the array. The proteins are then transcribed and translated by a cell-free system and immobilized in situ using epitope tags fused to the proteins. Sera are added, and bound IgG is detected by standard secondary reagents. Using a sequential screening strategy to select AAb from 4,988 candidate tumor antigens, we have identified 28 potential AAb biomarkers for the early detection of breast cancer, and here we propose to evaluate these biomarkers using the EDRN Breast Cancer Reference Set.

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

PubMed

Haas, Christian S; Creighton, Chad J; Pi, Xiujun; Maine, Ira; Koch, Alisa E; Haines, G Kenneth; Ling, Song; Chinnaiyan, Arul M; Holoshitz, Joseph

2006-07-01

To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

Microarray-based IgE detection in tears of patients with vernal keratoconjunctivitis.

PubMed

Leonardi, Andrea; Borghesan, Franco; Faggian, Diego; Plebani, Mario

2015-11-01

A specific allergen sensitization can be demonstrated in approximately half of the vernal keratoconjunctivitis (VKC) patients by conventional allergic tests. The measurement of specific IgE in tears using a multiplex allergen microarray may offer advantages to identify local sensitization to a specific allergen. In spring-summer 2011, serum and tears samples were collected from 10 active VKC patients (three females, seven males) and 10 age-matched normal subjects. Skin prick test, symptoms score and full ophthalmological examination were performed. Specific serum and tear IgE were assayed using ImmunoCAP ISAC, a microarray containing 103 components derived from 47 allergens. Normal subjects resulted negative for the presence of specific IgE both in serum and in tears. Of the 10 VKC patients, six resulted positive to specific IgE in serum and/or tears. In three of these six patients, specific IgE was found positive only in tears. Cross-reactivity between specific markers was found in three patients. Grass, tree, mites, animal but also food allergen-specific IgE were found in tears. Conjunctival provocation test performed out of season confirmed the specific local conjunctival reactivity. Multiple specific IgE measurements with single protein allergens using a microarray technique in tear samples are a useful, simple and non-invasive diagnostic tool. ImmunoCAP ISAC detects allergen sensitization at component level and adds important information by defining both cross- and co-sensitization to a large variety of allergen molecules. The presence of specific IgE only in tears of VKC patients reinforces the concept of possible local sensitization. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genome analysis of Legionella pneumophila strains using a mixed-genome microarray.

PubMed

Euser, Sjoerd M; Nagelkerke, Nico J; Schuren, Frank; Jansen, Ruud; Den Boer, Jeroen W

2012-01-01

Legionella, the causative agent for Legionnaires' disease, is ubiquitous in both natural and man-made aquatic environments. The distribution of Legionella genotypes within clinical strains is significantly different from that found in environmental strains. Developing novel genotypic methods that offer the ability to distinguish clinical from environmental strains could help to focus on more relevant (virulent) Legionella species in control efforts. Mixed-genome microarray data can be used to perform a comparative-genome analysis of strain collections, and advanced statistical approaches, such as the Random Forest algorithm are available to process these data. Microarray analysis was performed on a collection of 222 Legionella pneumophila strains, which included patient-derived strains from notified cases in The Netherlands in the period 2002-2006 and the environmental strains that were collected during the source investigation for those patients within the Dutch National Legionella Outbreak Detection Programme. The Random Forest algorithm combined with a logistic regression model was used to select predictive markers and to construct a predictive model that could discriminate between strains from different origin: clinical or environmental. Four genetic markers were selected that correctly predicted 96% of the clinical strains and 66% of the environmental strains collected within the Dutch National Legionella Outbreak Detection Programme. The Random Forest algorithm is well suited for the development of prediction models that use mixed-genome microarray data to discriminate between Legionella strains from different origin. The identification of these predictive genetic markers could offer the possibility to identify virulence factors within the Legionella genome, which in the future may be implemented in the daily practice of controlling Legionella in the public health environment.

Microbial community analysis of an Alabama coastal salt marsh impacted by the Deepwater Horizon Oil Spill

NASA Astrophysics Data System (ADS)

Beazley, M. J.; Martinez, R.; Rajan, S.; Powell, J.; Piceno, Y.; Tom, L.; Andersen, G. L.; Hazen, T. C.; Van Nostrand, J. D.; Zhou, J.; Mortazavi, B.; Sobecky, P. A.

2011-12-01

Microbial community responses of an Alabama coastal salt marsh environment to the Deepwater Horizon oil spill were studied by 16S rRNA (PhyloChip) and functional gene (GeoChip) microarray-based analysis. Oil and tar balls associated with the oil spill arrived along the Alabama coast in June 2010. Marsh and inlet sediment samples collected in June, July, and September 2010 from a salt marsh ecosystem at Point Aux Pines Alabama were analyzed to determine if bacterial community structure changed as a result of oil perturbation. Sediment total petroleum hydrocarbon (TPH) concentrations ranged from below detection to 189 mg kg-1 and were randomly dispersed throughout the salt marsh sediments. Total DNA extracted from sediment and particulates were used for PhyloChip and GeoChip hybridization. A total of 4000 to 8000 operational taxonomic units (OTUs) were detected in marsh and inlet samples. Distinctive changes in the number of detectable OTUs were observed between June, July, and September 2010. Surficial inlet sediments demonstrated a significant increase in the total number of OTUs between June and September that correlated with TPH concentrations. The most significant increases in bacterial abundance were observed in the phyla Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Verrucomicrobia. Bacterial richness in marsh sediments also correlated with TPH concentrations with significant changes primarily in Acidobacteria, Actinobacteria, Firmicutes, Fusobacteria, Nitrospirae, and Proteobacteria. GeoChip microarray analysis detected 5000 to 8300 functional genes in marsh and inlet samples. Surficial inlet sediments demonstrated distinctive increases in the number of detectable genes and gene signal intensities in July samples compared to June. Signal intensities increased (> 1.5-fold) in genes associated with petroleum degradation. Genes related to metal resistance, stress, and carbon cycling also demonstrated increases in oiled sediments. This study demonstrates the value of applying phylogenetic and functional gene microarray technology to characterize the extensive microbial diversity of marsh environments. Moreover, this technology provides significant insight into bacterial community responses to anthropogenic oil events.

Evaluation of the skin irritation using a DNA microarray on a reconstructed human epidermal model.

PubMed

Niwa, Makoto; Nagai, Kanji; Oike, Hideaki; Kobori, Masuko

2009-02-01

To avoid the need to use animals to test the skin irritancy potential of chemicals and cosmetics, it is important to establish an in vitro method based on the reconstructed human epidermal model. To evaluate skin irritancy efficiently and sensitively, we determined the gene expression induced by a topically-applied mild irritant sodium dodecyl sulfate (SDS) in a reconstructed human epidermal model LabCyte EPI-MODEL (LabCyte) using a DNA microarray carrying genes that were related to inflammation, immunity, stress and housekeeping. The expression and secretion of IL-1alpha in reconstructed human epidermal culture is known to be induced by irritation. We detected the induction of IL-1alpha expression and its secretion into the cell culture medium by treatment with 0.075% SDS for 18 h in LabCyte culture using DNA microarray, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and ELISA. DNA microarray analysis indicated that the expression of 10 of the 205 genes carried on the DNA microarray was significantly induced in a LabCyte culture by 0.05% or 0.075% SDS irritation for 18 h. RT-PCR analysis confirmed that SDS treatment significantly induced the expressions of interleukin-1 receptor antagonist (IL-1RN), FOS-like antigen 1 (FOSL1), heat shock 70 kDa protein 1A (HSPA1) and myeloid differentiation primary response gene (88) (MYD88), as well as the known marker genes for irritation IL-1beta and IL-8 in a LabCyte culture. Our results showed that a DNA microarray is a useful tool for efficiently evaluating mild skin irritation using a reconstructed human epidermal model.

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Optimized Probe Masking for Comparative Transcriptomics of Closely Related Species

PubMed Central

Poeschl, Yvonne; Delker, Carolin; Trenner, Jana; Ullrich, Kristian Karsten; Quint, Marcel; Grosse, Ivo

2013-01-01

Microarrays are commonly applied to study the transcriptome of specific species. However, many available microarrays are restricted to model organisms, and the design of custom microarrays for other species is often not feasible. Hence, transcriptomics approaches of non-model organisms as well as comparative transcriptomics studies among two or more species often make use of cost-intensive RNAseq studies or, alternatively, by hybridizing transcripts of a query species to a microarray of a closely related species. When analyzing these cross-species microarray expression data, differences in the transcriptome of the query species can cause problems, such as the following: (i) lower hybridization accuracy of probes due to mismatches or deletions, (ii) probes binding multiple transcripts of different genes, and (iii) probes binding transcripts of non-orthologous genes. So far, methods for (i) exist, but these neglect (ii) and (iii). Here, we propose an approach for comparative transcriptomics addressing problems (i) to (iii), which retains only transcript-specific probes binding transcripts of orthologous genes. We apply this approach to an Arabidopsis lyrata expression data set measured on a microarray designed for Arabidopsis thaliana, and compare it to two alternative approaches, a sequence-based approach and a genomic DNA hybridization-based approach. We investigate the number of retained probe sets, and we validate the resulting expression responses by qRT-PCR. We find that the proposed approach combines the benefit of sequence-based stringency and accuracy while allowing the expression analysis of much more genes than the alternative sequence-based approach. As an added benefit, the proposed approach requires probes to detect transcripts of orthologous genes only, which provides a superior base for biological interpretation of the measured expression responses. PMID:24260119

Development and Use of Integrated Microarray-Based Genomic Technologies for Assessing Microbial Community Composition and Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, J.; Wu, L.; Gentry, T.

2006-04-05

To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approximately 2000 unique and group-specific probes with <85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50 C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appearedmore » to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ({approx}1.3{sup o} 10{sup 7} cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r{sup 2} = 0.95-0.99). Application of this microarray to naphthalene-amended enrichments and soil microcosms demonstrated that composition of the microflora varied depending on incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in enrichments, the genes involved in naphthalene degradation from Gram-negative microorganisms such as Ralstonia, Comamonas, and Burkholderia were most abundant in the soil microcosms (as well as those for polyaromatic hydrocarbon and nitrotoluene degradation). Although naphthalene degradation is widely known and studied in Pseudomonas, Pseudomonas genes were not detected in either system. Real-time PCR analysis of 4 representative genes was consistent with microarray-based quantification (r{sup 2} = 0.95). Currently, we are also applying this microarray to the study of several different microbial communities and processes at the NABIR-FRC in Oak Ridge, TN. One project involves the monitoring of the development and dynamics of the microbial community of a fluidized bed reactor (FBR) used for reducing nitrate and the other project monitors microbial community responses to stimulation of uranium reducing populations via ethanol donor additions in situ and in a model system. Additionally, we are developing novel strategies for increasing microarray hybridization sensitivity. Finally, great improvements to our methods of probe design were made by the development of a new computer program, CommOligo. CommOligo designs unique and group-specific oligo probes for whole-genomes, metagenomes, and groups of environmental sequences and uses a new global alignment algorithm to design single or multiple probes for each gene or group. We are now using this program to design a more comprehensive functional gene array for environmental studies. Overall, our results indicate that the 50mer-based microarray technology has potential as a specific and quantitative tool to reveal the composition of microbial communities and their dynamics important to processes within contaminated environments.« less

Micro-array versus nano-array platforms: a comparative study for ODN detection based on SPR enhanced ellipsometry

NASA Astrophysics Data System (ADS)

Celen, Burcu; Demirel, Gökhan; Piskin, Erhan

2011-04-01

The rapid and sensitive detection of DNA has recently attracted worldwide attention for a variety of disease diagnoses and detection of harmful bacteria in food and drink. In this paper, we carried out a comparative study based on surface plasmon resonance enhanced ellipsometry (SPREE) for the detection of oligodeoxynucleotides (ODNs) using micro- and nano-array platforms. The micro-arrayed surfaces were fabricated by a photolithography approach using different types of mask having varying size and shape. Well-ordered arrays of high aspect ratio polymeric nanotubes were also obtained using high molecular weight polystyrene (PS) and anodic aluminum oxide (AAO) membranes having 200 nm pore diameters. The SPREE sensors were then prepared by direct coupling of thiolated probe-ODNs, which contain suitable spacer arms, on gold-coated micro- and nano-arrayed surfaces. We experimentally demonstrated that, for the first time, gold-coated free standing polymeric nano-arrayed platforms can easily be produced and lead to a significant sensor sensitivity gain compared to that of the conventional SPREE surfaces of about four times. We believe that such an enhancement in sensor response could be useful for next generation sensor systems.

Single-feature polymorphism discovery in the barley transcriptome

PubMed Central

Rostoks, Nils; Borevitz, Justin O; Hedley, Peter E; Russell, Joanne; Mudie, Sharon; Morris, Jenny; Cardle, Linda; Marshall, David F; Waugh, Robbie

2005-01-01

A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes. PMID:15960806

Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †

PubMed Central

La Duc, Myron T.; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J. A.; Venkateswaran, Kasthuri

2009-01-01

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments. PMID:19700540

Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides.

PubMed

Laassri, Majid; Chizhikov, Vladimir; Mikheev, Maxim; Shchelkunov, Sergei; Chumakov, Konstantin

2003-09-01

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

Design and verification of a pangenome microarray oligonucleotide probe set for Dehalococcoides spp.

PubMed

Hug, Laura A; Salehi, Maryam; Nuin, Paulo; Tillier, Elisabeth R; Edwards, Elizabeth A

2011-08-01

Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an oligonucleotide microarray probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome probe set was compared to probe sets designed independently for each of the five Dehalococcoides strains. The pangenome probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific probe sets on nontarget strains with <99% average nucleotide identity. An in silico analysis of the expected probe hybridization against the recently released Dehalococcoides strain GT genome and additional KB-1 metagenome sequence data indicated that the pangenome probe set performs more robustly than the combined strain-specific probe sets in the detection of genes not included in the original design. The pangenome probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined.

Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

PubMed

La Duc, Myron T; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J A; Venkateswaran, Kasthuri

2009-10-01

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.

HomSI: a homozygous stretch identifier from next-generation sequencing data.

PubMed

Görmez, Zeliha; Bakir-Gungor, Burcu; Sagiroglu, Mahmut Samil

2014-02-01

In consanguineous families, as a result of inheriting the same genomic segments through both parents, the individuals have stretches of their genomes that are homozygous. This situation leads to the prevalence of recessive diseases among the members of these families. Homozygosity mapping is based on this observation, and in consanguineous families, several recessive disease genes have been discovered with the help of this technique. The researchers typically use single nucleotide polymorphism arrays to determine the homozygous regions and then search for the disease gene by sequencing the genes within this candidate disease loci. Recently, the advent of next-generation sequencing enables the concurrent identification of homozygous regions and the detection of mutations relevant for diagnosis, using data from a single sequencing experiment. In this respect, we have developed a novel tool that identifies homozygous regions using deep sequence data. Using *.vcf (variant call format) files as an input file, our program identifies the majority of homozygous regions found by microarray single nucleotide polymorphism genotype data. HomSI software is freely available at www.igbam.bilgem.tubitak.gov.tr/softwares/HomSI, with an online manual.

High-resolution chromosomal microarrays in prenatal diagnosis significantly increase diagnostic power.

PubMed

Oneda, Beatrice; Baldinger, Rosa; Reissmann, Regina; Reshetnikova, Irina; Krejci, Pavel; Masood, Rahim; Ochsenbein-Kölble, Nicole; Bartholdi, Deborah; Steindl, Katharina; Morotti, Denise; Faranda, Marzia; Baumer, Alessandra; Asadollahi, Reza; Joset, Pascal; Niedrist, Dunja; Breymann, Christian; Hebisch, Gundula; Hüsler, Margaret; Mueller, René; Prentl, Elke; Wisser, Josef; Zimmermann, Roland; Rauch, Anita

2014-06-01

The objective of this study was to determine for the first time the reliability and the diagnostic power of high-resolution microarray testing in routine prenatal diagnostics. We applied high-resolution chromosomal microarray testing in 464 cytogenetically normal prenatal samples with any indication for invasive testing. High-resolution testing revealed a diagnostic yield of 6.9% and 1.6% in cases of fetal ultrasound anomalies and cases of advanced maternal age (AMA), respectively, which is similar to previous studies using low-resolution microarrays. In three (0.6%) additional cases with an indication of AMA, an aberration in susceptibility risk loci was detected. Moreover, one case (0.2%) showed an X-linked aberration in a female fetus, a finding relevant for future family planning. We found the rate of cases, in which the parents had to be tested for interpretation of unreported copy number variants (3.7%), and the rate of remaining variants of unknown significance (0.4%) acceptably low. Of note, these findings did not cause termination of pregnancy after expert genetic counseling. The 0.4% rate of confined placental mosaicism was similar to that observed by conventional karyotyping and notably involved a case of placental microdeletion. High-resolution prenatal microarray testing is a reliable technique that increases diagnostic yield by at least 17.3% when compared with conventional karyotyping, without an increase in the frequency of variants of uncertain significance. © 2014 John Wiley & Sons, Ltd.

Microarray assessment of virulence, antibiotic, and heavy metal resistance in an agricultural watershed creek.

PubMed

Unc, Adrian; Zurek, Ludek; Peterson, Greg; Narayanan, Sanjeev; Springthorpe, Susan V; Sattar, Syed A

2012-01-01

Potential risks associated with impaired surface water quality have commonly been evaluated by indirect description of potential sources using various fecal microbial indicators and derived source-tracking methods. These approaches are valuable for assessing and monitoring the impacts of land-use changes and changes in management practices at the source of contamination. A more detailed evaluation of putative etiologically significant genetic determinants can add value to these assessments. We evaluated the utility of using a microarray that integrates virulence genes with antibiotic and heavy metal resistance genes to describe and discriminate among spatially and seasonally distinct water samples from an agricultural watershed creek in Eastern Ontario. Because microarray signals may be analyzed as binomial distributions, the significance of ambiguous signals can be easily evaluated by using available off-the-shelf software. The FAMD software was used to evaluate uncertainties in the signal data. Analysis of multilocus fingerprinting data sets containing missing data has shown that, for the tested system, any variability in microarray signals had a marginal effect on data interpretation. For the tested watershed, results suggest that in general the wet fall season increased the downstream detection of virulence and resistance genes. Thus, the tested microarray technique has the potential to rapidly describe the quality of surface waters and thus to provide a qualitative tool to augment quantitative microbial risk assessments. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer.

PubMed

Beyer, Sasha J; Zhang, Xiaoli; Jimenez, Rafael E; Lee, Mei-Ling T; Richardson, Andrea L; Huang, Kun; Jhiang, Sissy M

2011-10-11

Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow

PubMed Central

Berry, Nadine Kaye; Bain, Nicole L; Enjeti, Anoop K; Rowlings, Philip

2014-01-01

Aim To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). Methods A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Results Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. Conclusions We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH. PMID:23969274

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow.

PubMed

Berry, Nadine Kaye; Bain, Nicole L; Enjeti, Anoop K; Rowlings, Philip

2014-01-01

To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

PubMed Central

Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

2016-01-01

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis. PMID:26493868

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

PubMed Central

Kamalakaran, Sitharthan; Kendall, Jude; Zhao, Xiaoyue; Tang, Chunlao; Khan, Sohail; Ravi, Kandasamy; Auletta, Theresa; Riggs, Michael; Wang, Yun; Helland, Åslaug; Naume, Bjørn; Dimitrova, Nevenka; Børresen-Dale, Anne-Lise; Hicks, Jim; Lucito, Robert

2009-01-01

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species. PMID:19474344

Fitting new technologies into the safety paradigm: use of microarrays in transfusion.

PubMed

Fournier-Wirth, C; Coste, J

2007-01-01

Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. The recent emergence of Nucleic acid Amplification Technologies (NAT) has revolutionised viral diagnosis, not only by increasing the level of sensitivity but also by facilitating the detection of several viruses in parallel by multiplexing specific primers. In more complex biological situations, when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems, such as DNA Arrays, permit a conceptually new approach. These miniaturised micro systems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, a reduction in the number of confirmation tests and a simplification of data interpretation. However, the systems currently available require additional instrumentation and reagents for sample preparation and target amplification prior to detection on the DNA array. A major challenge in the area of DNA detection is the development of methods that do not rely on target amplification systems. Likewise, the advances of protein microarrays have lagged because of poor stability of proteins, complex coupling chemistry and weak detection signals. Emerging technologies like Biosensors and nano-particle based DNA or Protein Bio-Barcode Amplification Assays are promising diagnostic tools for a wide range of clinical applications, including blood donation screening.

The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis.

PubMed

Heekin, Andrew M; Guerrero, Felix D; Bendele, Kylie G; Saldivar, Leo; Scoles, Glen A; Dowd, Scot E; Gondro, Cedric; Nene, Vishvanath; Djikeng, Appolinaire; Brayton, Kelly A

2013-09-23

Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.

The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis

PubMed Central

2013-01-01

Background Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. Methods A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. Results The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. Conclusion Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite. PMID:24330595

Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis.

PubMed

Valentini, Davide; Ferrara, Giovanni; Advani, Reza; Hallander, Hans O; Maeurer, Markus J

2015-07-01

Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355). Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes. 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines. We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.

TIPMaP: a web server to establish transcript isoform profiles from reliable microarray probes.

PubMed

Chitturi, Neelima; Balagannavar, Govindkumar; Chandrashekar, Darshan S; Abinaya, Sadashivam; Srini, Vasan S; Acharya, Kshitish K

2013-12-27

Standard 3' Affymetrix gene expression arrays have contributed a significantly higher volume of existing gene expression data than other microarray platforms. These arrays were designed to identify differentially expressed genes, but not their alternatively spliced transcript forms. No resource can currently identify expression pattern of specific mRNA forms using these microarray data, even though it is possible to do this. We report a web server for expression profiling of alternatively spliced transcripts using microarray data sets from 31 standard 3' Affymetrix arrays for human, mouse and rat species. The tool has been experimentally validated for mRNAs transcribed or not-detected in a human disease condition (non-obstructive azoospermia, a male infertility condition). About 4000 gene expression datasets were downloaded from a public repository. 'Good probes' with complete coverage and identity to latest reference transcript sequences were first identified. Using them, 'Transcript specific probe-clusters' were derived for each platform and used to identify expression status of possible transcripts. The web server can lead the user to datasets corresponding to specific tissues, conditions via identifiers of the microarray studies or hybridizations, keywords, official gene symbols or reference transcript identifiers. It can identify, in the tissues and conditions of interest, about 40% of known transcripts as 'transcribed', 'not-detected' or 'differentially regulated'. Corresponding additional information for probes, genes, transcripts and proteins can be viewed too. We identified the expression of transcripts in a specific clinical condition and validated a few of these transcripts by experiments (using reverse transcription followed by polymerase chain reaction). The experimental observations indicated higher agreements with the web server results, than contradictions. The tool is accessible at http://resource.ibab.ac.in/TIPMaP. The newly developed online tool forms a reliable means for identification of alternatively spliced transcript-isoforms that may be differentially expressed in various tissues, cell types or physiological conditions. Thus, by making better use of existing data, TIPMaP avoids the dependence on precious tissue-samples, in experiments with a goal to establish expression profiles of alternative splice forms--at least in some cases.

High quality epoxysilane substrate for clinical multiplex serodiagnostic proteomic microarrays

NASA Astrophysics Data System (ADS)

Ewart, Tom; Carmichael, Stuart; Lea, Peter

2005-09-01

Polylysine and aminopropylsilane treated glass comprised the majority of substrates employed in first generation genetic microarray substrates. Second generation single stranded long oligo libraries with amino termini provided for controlled terminal specific attachment, and rationally designed unique sequence libraries with normalized melting temperatures. These libraries benefit from active covalent coupling surfaces such as Epoxysilane. The latter's oxime ring shows versatile reactivity with amino-, thiol- and hydroxyl- groups thus encompassing small molecule, oligo and proteomic microarray applications. Batch-to-batch production uniformity supports entry of the Epoxysilane process into clinical diagnostics. We carried out multiple print runs of 21 clinically relevant bacterial and viral antigens at optimized concentrations, plus human IgG and IgM standards in triplicate on multiple batches of Epoxysilane substrates. A set of 45 patient sera were assayed in a 35 minute protocol using 10 microliters per array in a capillary-fill format (15 minute serum incubation, wash, 15 minute incubation with Cy3-labeled anti-hIgG plus Dy647-labeled anti-hIgM, final wash). The LOD (3 SD above background) was better than 1 microgram/ml for IgG, and standard curves were regular and monotonically increasing over the range 0 to 1000 micrograms/ml. Ninety-five percent of the CVs for the standards were under 10%, and 90% percent of CVs for antigen responses were under 10% across all batches of Epoxysilane and print runs. In addition, where SDs are larger than expected, microarray images may be readily reviewed for quality control purposes and pin misprints quickly identified. In order to determine the influence of stirring on sensitivity and speed of the microarray assay, we printed 10 common ToRCH antigens (H. pylori, T. gondii, Rubella, Rubeola, C. trachomatis, Herpes 1 and 2, CMV, C. jejuni, and EBV) in Epoxysilane-activated slide-wells. Anti-IgG-Cy3 direct binding to printed IgG calibration spots could be detected (3 x LOD) above background at 100 pg/ml (0.13 femtomoles sample content) in a 10 minute incubation. The LOD for detection of serum anti-H. pylori antibody level was 9 ng/ml in the same incubation time.

Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3.

PubMed

Lengger, Sandra; Otto, Johannes; Elsässer, Dennis; Schneider, Oliver; Tiehm, Andreas; Fleischer, Jens; Niessner, Reinhard; Seidel, Michael

2014-05-01

Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4-4.5 h. The parallel quantification was possible in a concentration range of 9.6 × 10(5)-1.4 × 10(10) genomic units (GU)/mL for bacteriophage MS2, 1.4 × 10(5)-3.7 × 10(8) GU/mL for bacteriophage ΦX174, and 6.5 × 10(3)-1.2 × 10(5) for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6 × 10(5) GU/mL for MS2, 5.3 × 10(3) GU/mL for ΦX174, and 1.5 × 10(2) GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.

Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.

PubMed

Zhang, Linsheng; Znoyko, Iya; Costa, Luciano J; Conlin, Laura K; Daber, Robert D; Self, Sally E; Wolff, Daynna J

2011-12-01

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. The methods currently used for monitoring CLL and determining conditions for treatment are limited in their ability to predict disease progression, patient survival, and response to therapy. Although clonal diversity and the acquisition of new chromosomal abnormalities during the disease course (clonal evolution) have been associated with disease progression, their prognostic potential has been underappreciated because cytogenetic and fluorescence in situ hybridization (FISH) studies have a restricted ability to detect genomic abnormalities and clonal evolution. We hypothesized that whole genome analysis using high resolution single nucleotide polymorphism (SNP) microarrays would be useful to detect diversity and infer clonal evolution to offer prognostic information. In this study, we used the Infinium Omni1 BeadChip (Illumina, San Diego, CA) array for the analysis of genetic variation and percent mosaicism in 25 non-selected CLL patients to explore the prognostic value of the assessment of clonal diversity in patients with CLL. We calculated the percentage of mosaicism for each abnormality by applying a mathematical algorithm to the genotype frequency data and by manual determination using the Simulated DNA Copy Number (SiDCoN) tool, which was developed from a computer model of mosaicism. At least one genetic abnormality was identified in each case, and the SNP data was 98% concordant with FISH results. Clonal diversity, defined as the presence of two or more genetic abnormalities with differing percentages of mosaicism, was observed in 12 patients (48%), and the diversity correlated with the disease stage. Clonal diversity was present in most cases of advanced disease (Rai stages III and IV) or those with previous treatment, whereas 9 of 13 patients without detected clonal diversity were asymptomatic or clinically stable. In conclusion, SNP microarray studies with simultaneous evaluation of genomic alterations and mosaic distribution of clones can be used to assess apparent clonal evolution via analysis of clonal diversity. Since clonal evolution in CLL is strongly correlated with disease progression, whole genome SNP microarray analysis provides a new comprehensive and reliable prognostic tool for CLL patients. Copyright © 2011 Elsevier Inc. All rights reserved.

ONLINE WATER MONITORING UTILIZING AN AUTOMATED MICROARRAY BIOSENSOR INSTRUMENT - PHASE I

EPA Science Inventory

Constellation Technology Corporation (Constellation) proposes the use of an integrated recovery and detection system for online water supply monitoring.  The integrated system is designed to efficiently capture and recover pathogens such as bacteria, viruses, parasites, an...

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

PubMed

Yu, Xiaobo; LaBaer, Joshua

2015-05-01

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing.

PubMed

Goel, Meenal; Verma, Abhishek; Gupta, Shalini

2018-07-15

Microarray technology to isolate living cells using external fields is a facile way to do phenotypic analysis at the cellular level. We have used alternating current dielectrophoresis (AC-DEP) to drive the assembly of live pathogenic Salmonella typhi (S.typhi) and Escherichia coli (E.coli) bacteria into miniaturized single cell microarrays. The effects of voltage and frequency were optimized to identify the conditions for maximum cell capture which gave an entrapment efficiency of 90% in 60 min. The chip was used for calibration-free estimation of cellular loads in binary mixtures and further applied for rapid and enhanced testing of cell viability in the presence of drug via impedance spectroscopy. Our results using a model antimicrobial sushi peptide showed that the cell viability could be tested down to 5 μg/mL drug concentration under an hour, thus establishing the utility of our system for ultrafast and sensitive detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of microbial genome-probing microarrays using digital multiple displacement amplification of uncultivated microbial single cells.

PubMed

Chang, Ho-Won; Sung, Youlboong; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Kim, Min-Soo; Jeon, Che Ok; Bae, Jin-Woo

2008-08-15

A crucial problem in the use of previously developed genome-probing microarrays (GPM) has been the inability to use uncultivated bacterial genomes to take advantage of the high sensitivity and specificity of GPM in microbial detection and monitoring. We show here a method, digital multiple displacement amplification (MDA), to amplify and analyze various genomes obtained from single uncultivated bacterial cells. We used 15 genomes from key microbes involved in dichloromethane (DCM)-dechlorinating enrichment as microarray probes to uncover the bacterial population dynamics of samples without PCR amplification. Genomic DNA amplified from single cells originating from uncultured bacteria with 80.3-99.4% similarity to 16S rRNA genes of cultivated bacteria. The digital MDA-GPM method successfully monitored the dynamics of DCM-dechlorinating communities from different phases of enrichment status. Without a priori knowledge of microbial diversity, the digital MDA-GPM method could be designed to monitor most microbial populations in a given environmental sample.

High-Throughput Lectin Microarray-Based Analysis of Live Cell Surface Glycosylation

PubMed Central

Li, Yu; Tao, Sheng-ce; Zhu, Heng; Schneck, Jonathan P.

2011-01-01

Lectins, plant-derived glycan-binding proteins, have long been used to detect glycans on cell surfaces. However, the techniques used to characterize serum or cells have largely been limited to mass spectrometry, blots, flow cytometry, and immunohistochemistry. While these lectin-based approaches are well established and they can discriminate a limited number of sugar isomers by concurrently using a limited number of lectins, they are not amenable for adaptation to a high-throughput platform. Fortunately, given the commercial availability of lectins with a variety of glycan specificities, lectins can be printed on a glass substrate in a microarray format to profile accessible cell-surface glycans. This method is an inviting alternative for analysis of a broad range of glycans in a high-throughput fashion and has been demonstrated to be a feasible method of identifying binding-accessible cell surface glycosylation on living cells. The current unit presents a lectin-based microarray approach for analyzing cell surface glycosylation in a high-throughput fashion. PMID:21400689

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin-3 Inhibitor.

PubMed

Dion, Johann; Advedissian, Tamara; Storozhylova, Nataliya; Dahbi, Samir; Lambert, Annie; Deshayes, Frédérique; Viguier, Mireille; Tellier, Charles; Poirier, Françoise; Téletchéa, Stéphane; Dussouy, Christophe; Tateno, Hiroaki; Hirabayashi, Jun; Grandjean, Cyrille

2017-12-14

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3'-mono- and 2,3'-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2'-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical Analysis of Microarray Data with Replicated Spots: A Case Study with Synechococcus WH8102

PubMed Central

Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; Haaland, D. M.; Timlin, J. A.; Elbourne, L. D. H.; Palenik, B.; Paulsen, I. T.

2009-01-01

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition. PMID:19404483

Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

PubMed

Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

2010-01-01

The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

Ethical issues raised by genetic testing with oligonucleotide microarrays.

PubMed

Grody, Wayne W

2003-02-01

Because genes and alterations within them determine the identity, characteristics, and inheritance of every individual, the application of genetic science to humans has long been surrounded by apprehension, controversy, and real or perceived potential for abuse. Crude eugenics practices of the past now find a theoretical rebirth and transformation through the use of modern molecular genetic technologies for mutation detection, predictive and prenatal diagnosis, and, ultimately, gene replacement. The advent of oligonucleotide microarray analysis, in which hundreds or thousands of genes and mutations can be tested in parallel, offers tremendous promise for more accurate, sensitive, and efficient genetic testing. At the same time, however, this powerful technology dramatically increases the number and scope of ethical concerns accompanying each individual test request. This article considers the evolution and implications of these concerns, from the initial ordering of a microarray test by the physician to such issues as informed consent, privacy, confidentiality, clinical utility, discrimination, stigmatization, ethnic and population impact, and reimbursement.

Statistical Analysis of Microarray Data with Replicated Spots: A Case Study with Synechococcus WH8102

DOE PAGES

Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; ...

2009-01-01

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in partmore » to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition.« less

Gene expression profiles in the testis associated with testis-ova in adult Japanese medaka (Oryziaslatipes) exposed to 17α-ethinylestradiol.

PubMed

Hirakawa, Ikumi; Miyagawa, Shinichi; Katsu, Yoshinao; Kagami, Yoshihiro; Tatarazako, Norihisa; Kobayashi, Tohru; Kusano, Teruhiko; Mizutani, Takeshi; Ogino, Yukiko; Takeuchi, Takashi; Ohta, Yasuhiko; Iguchi, Taisen

2012-05-01

The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L(-1) of 17α-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L(-1) EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L(-1) EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L(-1) EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia

Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). Allmore » assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.« less

Molecular probes and microarrays for the detection of toxic algae in the genera Dinophysis and Phalacroma (Dinophyta).

PubMed

Edvardsen, Bente; Dittami, Simon M; Groben, René; Brubak, Sissel; Escalera, Laura; Rodríguez, Francisco; Reguera, Beatriz; Chen, Jixin; Medlin, Linda K

2013-10-01

Dinophysis and Phalacroma species containing diarrheic shellfish toxins and pectenotoxins occur in coastal temperate waters all year round and prevent the harvesting of mussels during several months each year in regions in Europe, Chile, Japan, and New Zealand. Toxicity varies among morphologically similar species, and a precise identification is needed for early warning systems. Molecular techniques using ribosomal DNA sequences offer a means to identify and detect precisely the potentially toxic species. We designed molecular probes targeting the 18S rDNA at the family and genus levels for Dinophysis and Phalacroma and at the species level for Dinophysis acuminata, Dinophysis acuta, and Dinophysis norvegica, the most commonly occurring, potentially toxic species of these genera in Western European waters. Dot blot hybridizations with polymerase chain reaction (PCR)-amplified rDNA from 17 microalgae were used to demonstrate probe specificity. The probes were modified along with other published fluorescence in situ hybridization and PCR probes and tested for a microarray platform within the MIDTAL project ( http://www.midtal.com ). The microarray was applied to field samples from Norway and Spain and compared to microscopic cell counts. These probes may be useful for early warning systems and monitoring and can also be used in population dynamic studies to distinguish species and life cycle stages, such as cysts, and their distribution in time and space.

Classification of mislabelled microarrays using robust sparse logistic regression.

PubMed

Bootkrajang, Jakramate; Kabán, Ata

2013-04-01

Previous studies reported that labelling errors are not uncommon in microarray datasets. In such cases, the training set may become misleading, and the ability of classifiers to make reliable inferences from the data is compromised. Yet, few methods are currently available in the bioinformatics literature to deal with this problem. The few existing methods focus on data cleansing alone, without reference to classification, and their performance crucially depends on some tuning parameters. In this article, we develop a new method to detect mislabelled arrays simultaneously with learning a sparse logistic regression classifier. Our method may be seen as a label-noise robust extension of the well-known and successful Bayesian logistic regression classifier. To account for possible mislabelling, we formulate a label-flipping process as part of the classifier. The regularization parameter is automatically set using Bayesian regularization, which not only saves the computation time that cross-validation would take, but also eliminates any unwanted effects of label noise when setting the regularization parameter. Extensive experiments with both synthetic data and real microarray datasets demonstrate that our approach is able to counter the bad effects of labelling errors in terms of predictive performance, it is effective at identifying marker genes and simultaneously it detects mislabelled arrays to high accuracy. The code is available from http://cs.bham.ac.uk/∼jxb008. Supplementary data are available at Bioinformatics online.

ModuleMiner - improved computational detection of cis-regulatory modules: are there different modes of gene regulation in embryonic development and adult tissues?

PubMed Central

Van Loo, Peter; Aerts, Stein; Thienpont, Bernard; De Moor, Bart; Moreau, Yves; Marynen, Peter

2008-01-01

We present ModuleMiner, a novel algorithm for computationally detecting cis-regulatory modules (CRMs) in a set of co-expressed genes. ModuleMiner outperforms other methods for CRM detection on benchmark data, and successfully detects CRMs in tissue-specific microarray clusters and in embryonic development gene sets. Interestingly, CRM predictions for differentiated tissues exhibit strong enrichment close to the transcription start site, whereas CRM predictions for embryonic development gene sets are depleted in this region. PMID:18394174

Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

PubMed

Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

2016-10-01

High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH 2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seasonal dynamics of freshwater pathogens as measured by microarray at Lake Sapanca, a drinking water source in the north-eastern part of Turkey.

PubMed

Akçaalan, Reyhan; Albay, Meric; Koker, Latife; Baudart, Julia; Guillebault, Delphine; Fischer, Sabine; Weigel, Wilfried; Medlin, Linda K

2017-12-22

Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project μAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.

Evaluating reproducibility of differential expression discoveries in microarray studies by considering correlated molecular changes.

PubMed

Zhang, Min; Zhang, Lin; Zou, Jinfeng; Yao, Chen; Xiao, Hui; Liu, Qing; Wang, Jing; Wang, Dong; Wang, Chenguang; Guo, Zheng

2009-07-01

According to current consistency metrics such as percentage of overlapping genes (POG), lists of differentially expressed genes (DEGs) detected from different microarray studies for a complex disease are often highly inconsistent. This irreproducibility problem also exists in other high-throughput post-genomic areas such as proteomics and metabolism. A complex disease is often characterized with many coordinated molecular changes, which should be considered when evaluating the reproducibility of discovery lists from different studies. We proposed metrics percentage of overlapping genes-related (POGR) and normalized POGR (nPOGR) to evaluate the consistency between two DEG lists for a complex disease, considering correlated molecular changes rather than only counting gene overlaps between the lists. Based on microarray datasets of three diseases, we showed that though the POG scores for DEG lists from different studies for each disease are extremely low, the POGR and nPOGR scores can be rather high, suggesting that the apparently inconsistent DEG lists may be highly reproducible in the sense that they are actually significantly correlated. Observing different discovery results for a disease by the POGR and nPOGR scores will obviously reduce the uncertainty of the microarray studies. The proposed metrics could also be applicable in many other high-throughput post-genomic areas.

Reconstructing the temporal ordering of biological samples using microarray data.

PubMed

Magwene, Paul M; Lizardi, Paul; Kim, Junhyong

2003-05-01

Accurate time series for biological processes are difficult to estimate due to problems of synchronization, temporal sampling and rate heterogeneity. Methods are needed that can utilize multi-dimensional data, such as those resulting from DNA microarray experiments, in order to reconstruct time series from unordered or poorly ordered sets of observations. We present a set of algorithms for estimating temporal orderings from unordered sets of sample elements. The techniques we describe are based on modifications of a minimum-spanning tree calculated from a weighted, undirected graph. We demonstrate the efficacy of our approach by applying these techniques to an artificial data set as well as several gene expression data sets derived from DNA microarray experiments. In addition to estimating orderings, the techniques we describe also provide useful heuristics for assessing relevant properties of sample datasets such as noise and sampling intensity, and we show how a data structure called a PQ-tree can be used to represent uncertainty in a reconstructed ordering. Academic implementations of the ordering algorithms are available as source code (in the programming language Python) on our web site, along with documentation on their use. The artificial 'jelly roll' data set upon which the algorithm was tested is also available from this web site. The publicly available gene expression data may be found at http://genome-www.stanford.edu/cellcycle/ and http://caulobacter.stanford.edu/CellCycle/.

GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor.

PubMed

Davis, Sean; Meltzer, Paul S

2007-07-15

Microarray technology has become a standard molecular biology tool. Experimental data have been generated on a huge number of organisms, tissue types, treatment conditions and disease states. The Gene Expression Omnibus (Barrett et al., 2005), developed by the National Center for Bioinformatics (NCBI) at the National Institutes of Health is a repository of nearly 140,000 gene expression experiments. The BioConductor project (Gentleman et al., 2004) is an open-source and open-development software project built in the R statistical programming environment (R Development core Team, 2005) for the analysis and comprehension of genomic data. The tools contained in the BioConductor project represent many state-of-the-art methods for the analysis of microarray and genomics data. We have developed a software tool that allows access to the wealth of information within GEO directly from BioConductor, eliminating many the formatting and parsing problems that have made such analyses labor-intensive in the past. The software, called GEOquery, effectively establishes a bridge between GEO and BioConductor. Easy access to GEO data from BioConductor will likely lead to new analyses of GEO data using novel and rigorous statistical and bioinformatic tools. Facilitating analyses and meta-analyses of microarray data will increase the efficiency with which biologically important conclusions can be drawn from published genomic data. GEOquery is available as part of the BioConductor project.

Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

DTIC Science & Technology

2009-08-11

Competing Interests: One of the contributing authors : Clark Tibbetts, is the Executive Vice President and Chief Technology Officer of Tessarae, LLC...Detection 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR (S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7...N/A 1021 ng No detection Sin nombre Bunyaviridae III 1021 ng Pulmonary syndrome hantavirus strain Convict Creek 107 1CCHFV = Crimean-Congo hemorrhagic

Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

PubMed Central

Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

2011-01-01

Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

PubMed Central

Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

2009-01-01

Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. PMID:19123946

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray

PubMed Central

2010-01-01

Background Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Results Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. Conclusion All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues. PMID:20964859

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, G.L.; He, Z.; DeSantis, T.Z.

Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogeneticmore » microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.« less

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.

PubMed

Fenart, Stéphane; Ndong, Yves-Placide Assoumou; Duarte, Jorge; Rivière, Nathalie; Wilmer, Jeroen; van Wuytswinkel, Olivier; Lucau, Anca; Cariou, Emmanuelle; Neutelings, Godfrey; Gutierrez, Laurent; Chabbert, Brigitte; Guillot, Xavier; Tavernier, Reynald; Hawkins, Simon; Thomasset, Brigitte

2010-10-21

Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.

Hybrid genetic algorithm-neural network: feature extraction for unpreprocessed microarray data.

PubMed

Tong, Dong Ling; Schierz, Amanda C

2011-09-01

Suitable techniques for microarray analysis have been widely researched, particularly for the study of marker genes expressed to a specific type of cancer. Most of the machine learning methods that have been applied to significant gene selection focus on the classification ability rather than the selection ability of the method. These methods also require the microarray data to be preprocessed before analysis takes place. The objective of this study is to develop a hybrid genetic algorithm-neural network (GANN) model that emphasises feature selection and can operate on unpreprocessed microarray data. The GANN is a hybrid model where the fitness value of the genetic algorithm (GA) is based upon the number of samples correctly labelled by a standard feedforward artificial neural network (ANN). The model is evaluated by using two benchmark microarray datasets with different array platforms and differing number of classes (a 2-class oligonucleotide microarray data for acute leukaemia and a 4-class complementary DNA (cDNA) microarray dataset for SRBCTs (small round blue cell tumours)). The underlying concept of the GANN algorithm is to select highly informative genes by co-evolving both the GA fitness function and the ANN weights at the same time. The novel GANN selected approximately 50% of the same genes as the original studies. This may indicate that these common genes are more biologically significant than other genes in the datasets. The remaining 50% of the significant genes identified were used to build predictive models and for both datasets, the models based on the set of genes extracted by the GANN method produced more accurate results. The results also suggest that the GANN method not only can detect genes that are exclusively associated with a single cancer type but can also explore the genes that are differentially expressed in multiple cancer types. The results show that the GANN model has successfully extracted statistically significant genes from the unpreprocessed microarray data as well as extracting known biologically significant genes. We also show that assessing the biological significance of genes based on classification accuracy may be misleading and though the GANN's set of extra genes prove to be more statistically significant than those selected by other methods, a biological assessment of these genes is highly recommended to confirm their functionality. Copyright © 2011 Elsevier B.V. All rights reserved.

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

PubMed

Elsholz, B; Wörl, R; Blohm, L; Albers, J; Feucht, H; Grunwald, T; Jürgen, B; Schweder, T; Hintsche, Rainer

2006-07-15

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

The Changes of Gene Expression on Human Hair during Long-Spaceflight

NASA Astrophysics Data System (ADS)

Terada, Masahiro; Mukai, Chiaki; Ishioka, Noriaki; Majima, Hideyuki J.; Yamada, Shin; Seki, Masaya; Takahashi, Rika; Higashibata, Akira; Ohshima, Hiroshi; Sudoh, Masamichi; Minamisawa, Susumu

Hair has many advantages as the experimental sample. In a hair follicle, hair matrix cells actively divide and these active changes sensitively reflect physical condition on human body. The hair shaft records the metabolic conditions of mineral elements in our body. From human hairs, we can detect physiological informations about the human health. Therefore, we focused on using hair root analysis to understand the effects of spaceflight on astronauts. In 2009, we started a research program focusing on the analysis of astronauts’ hairs to examine the effects of long-term spaceflight on the gene expression in the human body. We want to get basic information to invent the effectivly diagnostic methods to detect the health situations of astronauts during space flight by analyzing human hair. We extracted RNA form the collected samples. Then, these extracted RNA was amplified. Amplified RNA was processed and hybridized to the Whole Human Genome (4×44K) Oligo Microarray (Agilent Technologies) according to the manufacturer’s protocol. Slide scanning was performed using the Agilent DNA Microarray Scanner. Scanning data were normalized with Agilent’s Feature Extraction software. Data preprocessing and analysis were performed using GeneSpring software 11.0.1. Next, Synthesis of cDNA (1 mg) was carried out using the PrimeScript RT reagent Kit (TaKaRa Bio) following the manufacturer’s instructions. The qRT-PCR experiment was performed with SYBR Premix Ex Taq (TaKaRa Bio) using the 7500 Real-Time PCR system (Applied Biosystems). We detected the changes of some gene expressions during spaceflight from both microarray and qRT-PCR data. These genes seems to be related with the hair proliferation. We believe that these results will lead to the discovery of the important factor effected during space flight on the hair.

[Clinical value of genome-wide high resolution chromosomal microarray analysis in etiological study of fetuses with congenital heart defects].

PubMed

Wu, Xiaoli; Fu, Fang; Li, Ru; Pan, Min; Han, Jin; Zhen, Li; Yang, Xin; Zhang, Yongling; Li, Fatao; Liao, Can

2014-12-01

To explore the clinical value of genome-wide high resolution chromosomal microarray analysis (CMA) in etiological study of fetuses with congenital heart disease (CHD) diagnosed by fetal echocardiography. A total of 176 fetuses diagnosed CHD by fetal echocardiography were analyzed, and invasive prenatal diagnosis was performed at Guangzhou Women and Children's Medical Center from January 2012 to January 2014. Among them, 158 fetuses were proved to have normal karyotype, and 88 fetuses (50.0%, 88/176) underwent CMA testing. The parental blood specimens were also collected for assisting the diagnosis of variants of uncertain clinical significance (VOUS). The 88 fetuses were divided into two groups: isolated CHD (n = 68) and CHD with extra-cardiac structural abnormalities (n = 20). The phenotypes of the two groups were subclassified. Copy number variations (CNV) were classified as benign CNV, pathogenic CNV (pCNV) or VOUS. (1) 58 fetuses (66%, 58/88) were with simple CHD and 30 fetuses were with complicated CHD (34%, 30/88). In the 45 fetuses with isolated and simple CHD, the pCNV detection rate was 11% (5/45). In the 23 fetuses with isolated and complicated CHD, the pCNV detection rate was 17% (4/23). In the 13 fetuses with simple CHD and extra-cardiac structural abnormalities, the pCNV detection rate was 5/13. In the 7 fetuses with complicated CHD and extra-cardiac structural abnormalities, the pCNV detection rate was 0. (2) The total detection rate for pCNV detection was 16% (14/88) in the 88 fetuses. The pCNV detection rates for isolated CHD and CHD with extra-cardiac structural abnormalities were 13% (9/68) and 25% (5/20), respectively (P > 0.05). The pCNV detection rates for simple and complicated CHD were 17% (10/58) and 13% (4/30), respectively (P > 0.05). (3) Eighteen fetuses (10.2%, 18/176) had abnormal karyotype results. (4) CMA test was performed in 88 fetuses. CNV detected in 8 fetuses were classified as VOUS initially. After parental microarray analysis, CNV in 5 fetuses were inherited and interpreted as benign. CNV in the other 3 fetuses (3%, 3/88) were remained unknown significance. CNV in 14 fetuses (16% ) were interpreted as pCNV. In fetuses with CHD and normal karyotype, the application of CMA could increase the detection rate of pCNV. Genome-wide CMA could be used as a regular tool in the prenatal diagnosis of fetuses with CHD and normal karyotype. This technology may benefit evaluation of fetal prognosis in prenatal genetic counselling.

puma: a Bioconductor package for propagating uncertainty in microarray analysis.

PubMed

Pearson, Richard D; Liu, Xuejun; Sanguinetti, Guido; Milo, Marta; Lawrence, Neil D; Rattray, Magnus

2009-07-09

Most analyses of microarray data are based on point estimates of expression levels and ignore the uncertainty of such estimates. By determining uncertainties from Affymetrix GeneChip data and propagating these uncertainties to downstream analyses it has been shown that we can improve results of differential expression detection, principal component analysis and clustering. Previously, implementations of these uncertainty propagation methods have only been available as separate packages, written in different languages. Previous implementations have also suffered from being very costly to compute, and in the case of differential expression detection, have been limited in the experimental designs to which they can be applied. puma is a Bioconductor package incorporating a suite of analysis methods for use on Affymetrix GeneChip data. puma extends the differential expression detection methods of previous work from the 2-class case to the multi-factorial case. puma can be used to automatically create design and contrast matrices for typical experimental designs, which can be used both within the package itself but also in other Bioconductor packages. The implementation of differential expression detection methods has been parallelised leading to significant decreases in processing time on a range of computer architectures. puma incorporates the first R implementation of an uncertainty propagation version of principal component analysis, and an implementation of a clustering method based on uncertainty propagation. All of these techniques are brought together in a single, easy-to-use package with clear, task-based documentation. For the first time, the puma package makes a suite of uncertainty propagation methods available to a general audience. These methods can be used to improve results from more traditional analyses of microarray data. puma also offers improvements in terms of scope and speed of execution over previously available methods. puma is recommended for anyone working with the Affymetrix GeneChip platform for gene expression analysis and can also be applied more generally.

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls

PubMed Central

2013-01-01

Background Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n = 4) or suffering from DCM (n = 4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p < 0.05 and fold change (FC) > 1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p > 0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p > 0.05). Conclusions Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases. PMID:23327631

Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls.

PubMed

Steudemann, Carola; Bauersachs, Stefan; Weber, Karin; Wess, Gerhard

2013-01-17

Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n=4) or suffering from DCM (n=4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p<0.05 and fold change (FC)>1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p>0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p>0.05). Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.

Chromosomal microarray analysis of Bulgarian patients with epilepsy and intellectual disability.

PubMed

Peycheva, Valentina; Kamenarova, Kunka; Ivanova, Neviana; Stamatov, Dimitar; Avdjieva-Tzavella, Daniela; Alexandrova, Iliana; Zhelyazkova, Sashka; Pacheva, Iliana; Dimova, Petya; Ivanov, Ivan; Litvinenko, Ivan; Bozhinova, Veneta; Tournev, Ivailo; Simeonov, Emil; Mitev, Vanyo; Jordanova, Albena; Kaneva, Radka

2018-08-15

High resolution chromosomal microarray analysis (CMA) has facilitated the identification of small chromosomal rearrangements throughout the genome, associated with various neurodevelopmental phenotypes, including ID/DD. Recently, it became evident that intellectual disability (ID)/developmental delay (DD) can occur with associated co-morbidities like epileptic seizures, autism and additional congenital anomalies. These observations require whole genome approach in order to detect the genetic causes of these complex disorders. In this study, we examined 92 patients of Bulgarian origin at age between 1 and 22 years with ID, generalized epilepsy, autistic signs and congenital anomalies. CMA was carried out using SurePrint G3 Human CGH Microarray Kit, 4 × 180 K and SurePrint G3 Unrestricted CGH ISCA v2, 4 × 180 K oligo platforms. Referral indications for selection of the patients were the presence of generalized refractory seizures disorders and co-morbid ID. Clearly pathogenic copy number variations (CNVs) were detected in eight patients (8.7%) from our cohort. Additionally, possibly pathogenic rearrangements of unclear clinical significance were detected in six individuals (6.5%), which make for an overall diagnostic yield of 15.2% among our cohort of patients. We report here the patients with clearly pathogenic CNVs, discuss the potential causality of the possibly pathogenic CNVs and make genotype - phenotype correlations. One novel possibly pathogenic heterozygous deletion in 15q22.31 region was detected in a case with ID/DD. Additionally, whole APBA2 gene duplication in 15q13.1 was found in three generations of a family with epilepsy, ID and psychiatric abnormalities. The results from this study allow us to define the genetic diagnosis in a subset of Bulgarian patients and improve the genetic counseling of the affected families. To our knowledge, this is the first aCGH evaluation of a Bulgarian cohort of children with epilepsy and ID so far. Copyright © 2018 Elsevier B.V. All rights reserved.

Sparse representation and Bayesian detection of genome copy number alterations from microarray data.

PubMed

Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C; Triche, Timothy J; Asgharzadeh, Shahab

2008-02-01

Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). http://biron.usc.edu/~piquereg/GADA

Application of an oligonucleotide microarray-based nano-amplification technique for the detection of fungal pathogens.

PubMed

Lu, Weiping; Gu, Dayong; Chen, Xingyun; Xiong, Renping; Liu, Ping; Yang, Nan; Zhou, Yuanguo

2010-10-01

The traditional techniques for diagnosis of invasive fungal infections in the clinical microbiology laboratory need improvement. These techniques are prone to delay results due to their time-consuming process, or result in misidentification of the fungus due to low sensitivity or low specificity. The aim of this study was to develop a method for the rapid detection and identification of fungal pathogens. The internal transcribed spacer two fragments of fungal ribosomal DNA were amplified using a polymerase chain reaction for all samples. Next, the products were hybridized with the probes immobilized on the surface of a microarray. These species-specific probes were designed to detect nine different clinical pathogenic fungi including Candida albicans, Candida tropocalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida guilliermondii, Candida keyfr, and Cryptococcus neoformans. The hybridizing signals were enhanced with gold nanoparticles and silver deposition, and detected using a flatbed scanner or visually. Fifty-nine strains of fungal pathogens, including standard and clinically isolated strains, were correctly identified by this method. The sensitivity of the assay for Candida albicans was 10 cells/mL. Ten cultures from clinical specimens and 12 clinical samples spiked with fungi were also identified correctly. This technique offers a reliable alternative to conventional methods for the detection and identification of fungal pathogens. It has higher efficiency, specificity and sensitivity compared with other methods commonly used in the clinical laboratory.

PAX3 gene deletion detected by microarray analysis in a girl with hearing loss.

PubMed

Drozniewska, Malgorzata; Haus, Olga

2014-01-01

Deletions of the PAX3 gene have been rarely reported in the literature. Mutations of this gene are a common cause of Waardenburg syndrome type 1 and 3. We report a 16 year old female presenting hearing loss and normal intellectual development, without major features of Waardenburg syndrome type 1, and without family history of the syndrome. Her phenotype, however, overlaps with features of craniofacial-deafness-hand syndrome. Microarray analysis showed ~862 kb de novo deletion at 2q36.1 including PAX3. The above findings suggest that the rearrangement found in our patient appeared de novo and with high probability is a cause of her phenotype.

Chromosomal Microarray Testing in 42 Korean Patients with Unexplained Developmental Delay, Intellectual Disability, Autism Spectrum Disorders, and Multiple Congenital Anomalies.

PubMed

Lee, Sun Ho; Song, Wung Joo

2017-09-01

Chromosomal microarray (CMA) is a high-resolution, high-throughput method of identifying submicroscopic genomic copy number variations (CNVs). CMA has been established as the first-line diagnostic test for individuals with developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), and multiple congenital anomalies (MCAs). CMA analysis was performed in 42 Korean patients who had been diagnosed with unexplained DD, ID, ASDs, and MCAs. Clinically relevant CNVs were discovered in 28 patients. Variants of unknown significance were detected in 13 patients. The diagnostic yield was high (66.7%). CMA is a superior diagnostic tool compared with conventional karyotyping and fluorescent in situ hybridization.

Flexible hemispheric microarrays of highly pressure-sensitive sensors based on breath figure method.

PubMed

Wang, Zhihui; Zhang, Ling; Liu, Jin; Jiang, Hao; Li, Chunzhong

2018-05-30

Recently, flexible pressure sensors featuring high sensitivity, broad sensing range and real-time detection have aroused great attention owing to their crucial role in the development of artificial intelligent devices and healthcare systems. Herein, highly sensitive pressure sensors based on hemisphere-microarray flexible substrates are fabricated via inversely templating honeycomb structures deriving from a facile and static breath figure process. The interlocked and subtle microstructures greatly improve the sensing characteristics and compressibility of the as-prepared pressure sensor, endowing it a sensitivity as high as 196 kPa-1 and a wide pressure sensing range (0-100 kPa), as well as other superior performance, including a lower detection limit of 0.5 Pa, fast response time (<26 ms) and high reversibility (>10 000 cycles). Based on the outstanding sensing performance, the potential capability of our pressure sensor in capturing physiological information and recognizing speech signals has been demonstrated, indicating promising application in wearable and intelligent electronics.

Mass-transport limitations in spot-based microarrays.

PubMed

Zhao, Ming; Wang, Xuefeng; Nolte, David

2010-09-20

Mass transport of analyte to surface-immobilized affinity reagents is the fundamental bottleneck for sensitive detection in solid-support microarrays and biosensors. Analyte depletion in the volume adjacent to the sensor causes deviation from ideal association, significantly slows down reaction kinetics, and causes inhomogeneous binding across the sensor surface. In this paper we use high-resolution molecular interferometric imaging (MI2), a label-free optical interferometry technique for direct detection of molecular films, to study the inhomogeneous distribution of intra-spot binding across 100 micron-diameter protein spots. By measuring intra-spot binding inhomogeneity, reaction kinetics can be determined accurately when combined with a numerical three-dimensional finite element model. To ensure homogeneous binding across a spot, a critical flow rate is identified in terms of the association rate k(a) and the spot diameter. The binding inhomogeneity across a spot can be used to distinguish high-affinity low-concentration specific reactions from low-affinity high-concentration non-specific binding of background proteins.

DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp.

PubMed

Schmoock, Gernot; Ehricht, Ralf; Melzer, Falk; Rassbach, Astrid; Scholz, Holger C; Neubauer, Heinrich; Sachse, Konrad; Mota, Rinaldo Aparecido; Saqib, Muhammad; Elschner, Mandy

2009-01-01

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.

Mixture models for detecting differentially expressed genes in microarrays.

PubMed

Jones, Liat Ben-Tovim; Bean, Richard; McLachlan, Geoffrey J; Zhu, Justin Xi

2006-10-01

An important and common problem in microarray experiments is the detection of genes that are differentially expressed in a given number of classes. As this problem concerns the selection of significant genes from a large pool of candidate genes, it needs to be carried out within the framework of multiple hypothesis testing. In this paper, we focus on the use of mixture models to handle the multiplicity issue. With this approach, a measure of the local FDR (false discovery rate) is provided for each gene. An attractive feature of the mixture model approach is that it provides a framework for the estimation of the prior probability that a gene is not differentially expressed, and this probability can subsequently be used in forming a decision rule. The rule can also be formed to take the false negative rate into account. We apply this approach to a well-known publicly available data set on breast cancer, and discuss our findings with reference to other approaches.

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection inmore » archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.« less

DFP: a Bioconductor package for fuzzy profile identification and gene reduction of microarray data

PubMed Central

Glez-Peña, Daniel; Álvarez, Rodrigo; Díaz, Fernando; Fdez-Riverola, Florentino

2009-01-01

Background Expression profiling assays done by using DNA microarray technology generate enormous data sets that are not amenable to simple analysis. The greatest challenge in maximizing the use of this huge amount of data is to develop algorithms to interpret and interconnect results from different genes under different conditions. In this context, fuzzy logic can provide a systematic and unbiased way to both (i) find biologically significant insights relating to meaningful genes, thereby removing the need for expert knowledge in preliminary steps of microarray data analyses and (ii) reduce the cost and complexity of later applied machine learning techniques being able to achieve interpretable models. Results DFP is a new Bioconductor R package that implements a method for discretizing and selecting differentially expressed genes based on the application of fuzzy logic. DFP takes advantage of fuzzy membership functions to assign linguistic labels to gene expression levels. The technique builds a reduced set of relevant genes (FP, Fuzzy Pattern) able to summarize and represent each underlying class (pathology). A last step constructs a biased set of genes (DFP, Discriminant Fuzzy Pattern) by intersecting existing fuzzy patterns in order to detect discriminative elements. In addition, the software provides new functions and visualisation tools that summarize achieved results and aid in the interpretation of differentially expressed genes from multiple microarray experiments. Conclusion DFP integrates with other packages of the Bioconductor project, uses common data structures and is accompanied by ample documentation. It has the advantage that its parameters are highly configurable, facilitating the discovery of biologically relevant connections between sets of genes belonging to different pathologies. This information makes it possible to automatically filter irrelevant genes thereby reducing the large volume of data supplied by microarray experiments. Based on these contributions GENECBR, a successful tool for cancer diagnosis using microarray datasets, has recently been released. PMID:19178723

DFP: a Bioconductor package for fuzzy profile identification and gene reduction of microarray data.

PubMed

Glez-Peña, Daniel; Alvarez, Rodrigo; Díaz, Fernando; Fdez-Riverola, Florentino

2009-01-29

Expression profiling assays done by using DNA microarray technology generate enormous data sets that are not amenable to simple analysis. The greatest challenge in maximizing the use of this huge amount of data is to develop algorithms to interpret and interconnect results from different genes under different conditions. In this context, fuzzy logic can provide a systematic and unbiased way to both (i) find biologically significant insights relating to meaningful genes, thereby removing the need for expert knowledge in preliminary steps of microarray data analyses and (ii) reduce the cost and complexity of later applied machine learning techniques being able to achieve interpretable models. DFP is a new Bioconductor R package that implements a method for discretizing and selecting differentially expressed genes based on the application of fuzzy logic. DFP takes advantage of fuzzy membership functions to assign linguistic labels to gene expression levels. The technique builds a reduced set of relevant genes (FP, Fuzzy Pattern) able to summarize and represent each underlying class (pathology). A last step constructs a biased set of genes (DFP, Discriminant Fuzzy Pattern) by intersecting existing fuzzy patterns in order to detect discriminative elements. In addition, the software provides new functions and visualisation tools that summarize achieved results and aid in the interpretation of differentially expressed genes from multiple microarray experiments. DFP integrates with other packages of the Bioconductor project, uses common data structures and is accompanied by ample documentation. It has the advantage that its parameters are highly configurable, facilitating the discovery of biologically relevant connections between sets of genes belonging to different pathologies. This information makes it possible to automatically filter irrelevant genes thereby reducing the large volume of data supplied by microarray experiments. Based on these contributions GENECBR, a successful tool for cancer diagnosis using microarray datasets, has recently been released.

Microarray data mining using Bioconductor packages.

PubMed

Nie, Haisheng; Neerincx, Pieter B T; van der Poel, Jan; Ferrari, Francesco; Bicciato, Silvio; Leunissen, Jack A M; Groenen, Martien A M

2009-07-16

This paper describes the results of a Gene Ontology (GO) term enrichment analysis of chicken microarray data using the Bioconductor packages. By checking the enriched GO terms in three contrasts, MM8-PM8, MM8-MA8, and MM8-MM24, of the provided microarray data during this workshop, this analysis aimed to investigate the host reactions in chickens occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. The results of GO enrichment analysis using GO terms annotated to chicken genes and GO terms annotated to chicken-human orthologous genes were also compared. Furthermore, a locally adaptive statistical procedure (LAP) was performed to test differentially expressed chromosomal regions, rather than individual genes, in the chicken genome after Eimeria challenge. GO enrichment analysis identified significant (raw p-value < 0.05) GO terms for all three contrasts included in the analysis. Some of the GO terms linked to, generally, primary immune responses or secondary immune responses indicating the GO enrichment analysis is a useful approach to analyze microarray data. The comparisons of GO enrichment results using chicken gene information and chicken-human orthologous gene information showed more refined GO terms related to immune responses when using chicken-human orthologous gene information, this suggests that using chicken-human orthologous gene information has higher power to detect significant GO terms with more refined functionality. Furthermore, three chromosome regions were identified to be significantly up-regulated in contrast MM8-PM8 (q-value < 0.01). Overall, this paper describes a practical approach to analyze microarray data in farm animals where the genome information is still incomplete. For farm animals, such as chicken, with currently limited gene annotation, borrowing gene annotation information from orthologous genes in well-annotated species, such as human, will help improve the pathway analysis results substantially. Furthermore, LAP analysis approach is a relatively new and very useful way to be applied in microarray analysis.

Development of a chip-based multiplexed immunoassay using liposomal nanovesicles and its application in the detection of pathogens causing female lower genital tract infections.

PubMed

Su, Wen-Hsiang; Ho, Tien-Yu; Tsou, Tsung-Shan; Lee, Wen-Ling; Wang, Kuan-Chin; Yu, Yuan-Yi; Chen, Tien-Jui; Tan, Chia-Hsuan; Kuo, Cheng-Deng; Chen, Chien-Sheng; Wang, Peng-Hui

2013-03-01

Cervicovaginitis is a highly prevalent disease that is a burden on healthcare globally. Immediate and adequate treatment can eradicate the infection and block subsequent complications. The feasibility of achip-based multiplexed immunoassay using liposomal nanovesicles was tested. A multiplexed immunoassay chip containing five antibodies for five pathogens (Chlamydia trachomatis, Escherichia coli, Neisseria gonorrhoeae, Streptococcus agalactiae, and Candida albicans) was established and tested. Four patients with spiking of candidiasis were enrolled. The difference between positive and negative readings was evaluated using the paired Student t test. The detection threshold of Candida in this microarray was 100,000 CFU/mL in a vaginal sample, and the time required for the whole procedure was 3 hours. The testing of the four patients showed 100% for both sensitivity and specificity. This microarray chip was a rapid, easy, inexpensive and sensitive tool for detecting female lower genital tract Candida infection in a one-time vaginal sampling process, although the data on the four other pathogens were still unavailable. A larger population study is encouraged to test the validity of this multiplexed immunoassay chip. Copyright © 2013. Published by Elsevier B.V.

cn.FARMS: a latent variable model to detect copy number variations in microarray data with a low false discovery rate.

PubMed

Clevert, Djork-Arné; Mitterecker, Andreas; Mayr, Andreas; Klambauer, Günter; Tuefferd, Marianne; De Bondt, An; Talloen, Willem; Göhlmann, Hinrich; Hochreiter, Sepp

2011-07-01

Cost-effective oligonucleotide genotyping arrays like the Affymetrix SNP 6.0 are still the predominant technique to measure DNA copy number variations (CNVs). However, CNV detection methods for microarrays overestimate both the number and the size of CNV regions and, consequently, suffer from a high false discovery rate (FDR). A high FDR means that many CNVs are wrongly detected and therefore not associated with a disease in a clinical study, though correction for multiple testing takes them into account and thereby decreases the study's discovery power. For controlling the FDR, we propose a probabilistic latent variable model, 'cn.FARMS', which is optimized by a Bayesian maximum a posteriori approach. cn.FARMS controls the FDR through the information gain of the posterior over the prior. The prior represents the null hypothesis of copy number 2 for all samples from which the posterior can only deviate by strong and consistent signals in the data. On HapMap data, cn.FARMS clearly outperformed the two most prevalent methods with respect to sensitivity and FDR. The software cn.FARMS is publicly available as a R package at http://www.bioinf.jku.at/software/cnfarms/cnfarms.html.

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.

PubMed

Chiba, Tetsuhiro; Kita, Kaoru; Zheng, Yun-Wen; Yokosuka, Osamu; Saisho, Hiromitsu; Iwama, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2006-07-01

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping.

PubMed

Jørgensen, Malene; Bæk, Rikke; Pedersen, Shona; Søndergaard, Evo K L; Kristensen, Søren R; Varming, Kim

2013-01-01

Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 10(4) cells was needed to obtain signals or that only 2.5×10(4) exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1-10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.