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Sample records for antibody microarray analysis

  1. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  2. Microarray Analysis of Antibodies Induced with Synthetic Antitumor Vaccines: Specificity against Diverse Mucin Core Structures.

    PubMed

    Pett, Christian; Cai, Hui; Liu, Jia; Palitzsch, Björn; Schorlemer, Manuel; Hartmann, Sebastian; Stergiou, Natascha; Lu, Mengji; Kunz, Horst; Schmitt, Edgar; Westerlind, Ulrika

    2017-03-17

    Glycoprotein research is pivotal for vaccine development and biomarker discovery. Many successful methodologies for reliably increasing the antigenicity toward tumor-associated glycopeptide structures have been reported. Deeper insights into the quality and specificity of the raised polyclonal, humoral reactions are often not addressed, despite the fact that an immunological memory, which produces antibodies with cross-reactivity to epitopes exposed on healthy cells, may cause autoimmune diseases. In the current work, three MUC1 antitumor vaccine candidates conjugated with different immune stimulants are evaluated immunologically. For assessment of the influence of the immune stimulant on antibody recognition, a comprehensive library of mucin 1 glycopeptides (>100 entries) is synthesized and employed in antibody microarray profiling; these range from small tumor-associated glycans (TN , STN , and T-antigen structures) to heavily extended O-glycan core structures (type-1 and type-2 elongated core 1-3 tri-, tetra-, and hexasaccharides) glycosylated in variable density at the five different sites of the MUC1 tandem repeat. This is one of the most extensive glycopeptide libraries ever made through total synthesis. On tumor cells, the core 2 β-1,6-N-acetylglucosaminyltransferase-1 (C2GlcNAcT-1) is down-regulated, resulting in lower amounts of the branched core 2 structures, which favor formation of linear core 1 or core 3 structures, and in particular, truncated tumor-associated antigen structures. The core 2 structures are commonly found on healthy cells and the elucidation of antibody cross-reactivity to such epitopes may predict the tumor-selectivity and safety of synthetic vaccines. With the extended mucin core structures in hand, antibody cross-reactivity toward the branched core 2 glycopeptide epitopes is explored. It is observed that the induced antibodies recognize MUC1 peptides with very high glycosylation site specificity. The nature of the

  3. Distinct antibody responses of patients with mild and severe leptospirosis determined by whole proteome microarray analysis

    PubMed Central

    Lessa-Aquino, Carolina; Lindow, Janet C.; Randall, Arlo; Wunder, Elsio; Pablo, Jozelyn; Nakajima, Rie; Jasinskas, Algis; Cruz, Jaqueline S.; Damião, Alcineia O.; Nery, Nívison; Ribeiro, Guilherme S.; Costa, Federico; Hagan, José E.; Reis, Mitermayer Galvão; Ko, Albert I.; Medeiros, Marco Alberto; Felgner, Philip L.

    2017-01-01

    Background Leptospirosis is an important zoonotic disease worldwide. Humans usually present a mild non-specific febrile illness, but a proportion of them develop more severe outcomes, such as multi-organ failure, lung hemorrhage and death. Such complications are thought to depend on several factors, including the host immunity. Protective immunity is associated with humoral immune response, but little is known about the immune response mounted during naturally-acquired Leptospira infection. Methods and principal findings Here, we used protein microarray chip to profile the antibody responses of patients with severe and mild leptospirosis against the complete Leptospira interrogans serovar Copenhageni predicted ORFeome. We discovered a limited number of immunodominant antigens, with 36 antigens specific to patients, of which 11 were potential serodiagnostic antigens, identified at acute phase, and 33 were potential subunit vaccine targets, detected after recovery. Moreover, we found distinct antibody profiles in patients with different clinical outcomes: in the severe group, overall IgM responses do not change and IgG responses increase over time, while both IgM and IgG responses remain stable in the mild patient group. Analyses of individual patients’ responses showed that >74% of patients in the severe group had significant IgG increases over time compared to 29% of patients in the mild group. Additionally, 90% of IgM responses did not change over time in the mild group, compared to ~51% in the severe group. Conclusions In the present study, we detected antibody profiles associated with disease severity and speculate that patients with mild disease were protected from severe outcomes due to pre-existing antibodies, while patients with severe leptospirosis demonstrated an antibody profile typical of first exposure. Our findings represent a significant advance in the understanding of the humoral immune response to Leptospira infection, and we have identified new

  4. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

    PubMed Central

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

    2017-01-01

    Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

  5. Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides.

    PubMed

    Heimburg-Molinaro, Jamie; Priest, Jeffrey W; Live, David; Boons, Geert-Jan; Song, Xuezheng; Cummings, Richard D; Mead, Jan R

    2013-10-01

    Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.

  6. Antibody microarrays for native toxin detection.

    PubMed

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E

    2005-04-15

    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  7. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    PubMed Central

    Gao, Wei-Min; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S; Haab, Brian B; Hanash, Samir M

    2005-01-01

    Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer. PMID:16117833

  8. Discrimination of Influenza Infection (A/2009 H1N1) from Prior Exposure by Antibody Protein Microarray Analysis

    PubMed Central

    te Beest, Dennis; de Bruin, Erwin; Imholz, Sandra; Wallinga, Jacco; Teunis, Peter; Koopmans, Marion; van Boven, Michiel

    2014-01-01

    Reliable discrimination of recent influenza A infection from previous exposure using hemagglutination inhibition (HI) or virus neutralization tests is currently not feasible. This is due to low sensitivity of the tests and the interference of antibody responses generated by previous infections. Here we investigate the diagnostic characteristics of a newly developed antibody (HA1) protein microarray using data from cross-sectional serological studies carried out before and after the pandemic of 2009. The data are analysed by mixture models, providing a probabilistic classification of sera (susceptible, prior-exposed, recently infected). Estimated sensitivity and specificity for identifying A/2009 infections are low using HI (66% and 51%), and high when using A/2009 microarray data alone or together with A/1918 microarray data (96% and 95%). As a heuristic, a high A/2009 to A/1918 antibody ratio (>1.05) is indicative of recent infection, while a low ratio is indicative of a pre-existing response, even if the A/2009 titer is high. We conclude that highly sensitive and specific classification of individual sera is possible using the protein microarray, thereby enabling precise estimation of age-specific infection attack rates in the population even if sample sizes are small. PMID:25405997

  9. Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

    DTIC Science & Technology

    2009-08-27

    aeruginosa, Salmonella ty- phimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies en- abled detection of...Antisera against Salmonella typhimurium (recognizing a Antibody Biomarkers for Plague Molecular & Cellular Proteomics 8.5 925 by on A ugust 27, 2009 w w...clinical review of 27 cases. Arch. Intern. Med. 152, 1253–1256 9. Nielsen, K., Smith, P., Yu, W. L., and Halbert, G. (2007) Salmonella enterica serotype

  10. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.

    PubMed

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J; Helmsing, Saskia; Meier, Doris; Hust, Michael; Schröder, Christoph; Bertinetti, Daniela; Winter, Gerhard; Pardes, Khalid; Funk, Mia; Vala, Andrea; Giese, Nathalia; Herberg, Friedrich W; Dübel, Stefan; Hoheisel, Jörg D

    2016-09-25

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis.

  11. The SOLID (Signs Of LIfe Detector) instruments, antibody microarray based biosensors for in situ analysis: environmental immuno-profiles as biosignatures

    NASA Astrophysics Data System (ADS)

    Parro, Víctor

    Up to now most of the techniques used for organics or life detection in space missions are based on the detection of volatiles compounds by gas chromatography mass spectrometry (GC-MS). This was the case for the Viking's and Cassini/Huygens missions, or for the proposed SAM instrument for MSL. Even the Urey instrument, proposed for ESA's ExoMars mission, which focus on the analysis of the fluorescent-tagged volatiles by capillary electrophoresis. Sandwich antibody microarray immnunoassay is an excellent technique for the detection of complex and non volatile biological polymers (Parro et al., Space Sci. Rev, 2007. DOI 10.1007/s11214- 007-9276-1). A positive result in a sandwich immunoassay indicates that the sample contains a relatively complex molecular structure with at least two antigenic determinants, otherwise sandwich could not be detected. We have reported (Rivas et al., submitted) a shotgun approach for antibody production for biomarker detection in astrobiology and environmental monitoring, the production and testing of 155 new polyclonal antibodies against different bacteria and natural samples (water, sediments, soil, biofilms, etc) from Mars analog environments, as well as the construction and validation of a Life Detector Chip (LDCHIP200) with more than 200 antibodies for monitoring the presence of such bacteria or some of their remains. Some of the antibodies produced against iron-sulfur rich Rio Tinto environment (SW Spain) reacted against biological polymers from samples taken around the world (Antarctica, Yellowstone, 4 km depth mine in South Africa or Iceland). 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

  12. Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

    PubMed

    McBride, Ryan; Head, Steven R; Ordoukhanian, Phillip; Law, Mansun

    2016-01-01

    With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.

  13. Microtiter plate-based antibody microarrays for bacteria and toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 (...

  14. Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

    DTIC Science & Technology

    2011-01-01

    Appli- cations of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnology Adv 27, 502–520. 6. Denzin , L. K...4-20. J Biol Chem 266, 14095–14103. Transfected Cell Microarrays 137 7. Denzin , L. K., Gulliver, G. A., Voss, E. W., Jr. (1993) Mutational analysis of

  15. Regeneration of recombinant antigen microarrays for the automated monitoring of antibodies against zoonotic pathogens in swine sera.

    PubMed

    Meyer, Verena K; Kober, Catharina; Niessner, Reinhard; Seidel, Michael

    2015-01-23

    The ability to regenerate immobilized proteins like recombinant antigens (rAgs) on surfaces is an unsolved problem for flow-based immunoassays on microarray analysis systems. The regeneration on microarray chip surfaces is achieved by changing the protein structures and desorption of antibodies. Afterwards, reactivation of immobilized protein antigens is necessary for reconstitution processes. Any backfolding should be managed in a way that antibodies are able to detect the protein antigens in the next measurement cycle. The regeneration of rAg microarrays was examined for the first time on the MCR3 flow-based chemiluminescence (CL) microarray analysis platform. The aim was to reuse rAg microarray chips in order to reduce the screening effort and costs. An antibody capturing format was used to detect antibodies against zoonotic pathogens in sera of slaughtered pigs. Different denaturation and reactivation buffers were tested. Acidic glycine-SDS buffer (pH 2.5) and 8 M guanidinium hydrochloride showed the best results in respect of denaturation efficiencies. The highest CL signals after regeneration were achieved with a carbonate buffer containing 10 mM DTT and 0.1% BSA for reactivation. Antibodies against Yersinia spp. and hepatitis E virus (HEV) were detected in swine sera on one immunochip over 4 days and 25 measurement cycles. Each cycle took 10 min for detection and regeneration. By using the rAg microarray chip, a fast and automated screening of antibodies against pathogens in sera of slaughtered pigs would be possible for zoonosis monitoring.

  16. Automated Microarray Image Analysis Toolbox for MATLAB

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Willse, Alan R.; Protic, Miroslava; Chandler, Darrell P.

    2005-09-01

    The Automated Microarray Image Analysis (AMIA) Toolbox for MATLAB is a flexible, open-source microarray image analysis tool that allows the user to customize analysis of sets of microarray images. This tool provides several methods of identifying and quantify spot statistics, as well as extensive diagnostic statistics and images to identify poor data quality or processing. The open nature of this software allows researchers to understand the algorithms used to provide intensity estimates and to modify them easily if desired.

  17. An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling

    PubMed Central

    Price, Jordan V.; Tabakman, Scott M.; Li, Yanguang; Gong, Ming; Hong, Guosong; Feng, Ju; Utz, Paul J.; Dai, Hongjie

    2013-01-01

    High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone. PMID:23923050

  18. Analysis of DNA microarray expression data.

    PubMed

    Simon, Richard

    2009-06-01

    DNA microarrays are powerful tools for studying biological mechanisms and for developing prognostic and predictive classifiers for identifying the patients who require treatment and are best candidates for specific treatments. Because microarrays produce so much data from each specimen, they offer great opportunities for discovery and great dangers or producing misleading claims. Microarray based studies require clear objectives for selecting cases and appropriate analysis methods. Effective analysis of microarray data, where the number of measured variables is orders of magnitude greater than the number of cases, requires specialized statistical methods which have recently been developed. Recent literature reviews indicate that serious problems of analysis exist a substantial proportion of publications. This manuscript attempts to provide a non-technical summary of the key principles of statistical design and analysis for studies that utilize microarray expression profiling.

  19. Mutational analysis using oligonucleotide microarrays

    PubMed Central

    Hacia, J.; Collins, F.

    1999-01-01

    The development of inexpensive high throughput methods to identify individual DNA sequence differences is important to the future growth of medical genetics. This has become increasingly apparent as epidemiologists, pathologists, and clinical geneticists focus more attention on the molecular basis of complex multifactorial diseases. Such undertakings will rely upon genetic maps based upon newly discovered, common, single nucleotide polymorphisms. Furthermore, candidate gene approaches used in identifying disease associated genes necessitate screening large sequence blocks for changes tracking with the disease state. Even after such genes are isolated, large scale mutational analyses will often be needed for risk assessment studies to define the likely medical consequences of carrying a mutated gene.
This review concentrates on the use of oligonucleotide arrays for hybridisation based comparative sequence analysis. Technological advances within the past decade have made it possible to apply this technology to many different aspects of medical genetics. These applications range from the detection and scoring of single nucleotide polymorphisms to mutational analysis of large genes. Although we discuss published scientific reports, unpublished work from the private sector12 could also significantly affect the future of this technology.


Keywords: mutational analysis; oligonucleotide microarrays; DNA chips PMID:10528850

  20. Optimizing scan parameters for antibody microarray experiments: accelerating robust systems diagnostics for life sciences.

    PubMed

    Gu, Qiang; Sivanandam, Thamil Mani

    2014-06-01

    Microarray experiments are a centerpiece of postgenomics life sciences and the current efforts to develop systems diagnostics for personalized medicine. The majority of antibody microarray experiments are fluorescence-based, which utilizes a scanner to convert target signals into image files for subsequent quantification. Certain scan parameters such as the laser power and photomultiplier tube gain (PMT) can influence the readout of fluorescent intensities and thus may affect data quantitation. To date, however, there is no consensus of how to determine the optimal settings of microarray scanners. Here we show that different settings of the laser power and PMT not only affect the signal intensities but also the accuracy of antibody microarray experiments. More importantly, we demonstrate an experimental approach using two fluorescent dyes to determine optimal settings of scan parameters for microarray experiments. These measures provide added quality control of microarray experiments, and thus help to improve the accuracy of quantitative outcome in microarray experiments in the above contexts.

  1. MARS: Microarray analysis, retrieval, and storage system

    PubMed Central

    Maurer, Michael; Molidor, Robert; Sturn, Alexander; Hartler, Juergen; Hackl, Hubert; Stocker, Gernot; Prokesch, Andreas; Scheideler, Marcel; Trajanoski, Zlatko

    2005-01-01

    Background Microarray analysis has become a widely used technique for the study of gene-expression patterns on a genomic scale. As more and more laboratories are adopting microarray technology, there is a need for powerful and easy to use microarray databases facilitating array fabrication, labeling, hybridization, and data analysis. The wealth of data generated by this high throughput approach renders adequate database and analysis tools crucial for the pursuit of insights into the transcriptomic behavior of cells. Results MARS (Microarray Analysis and Retrieval System) provides a comprehensive MIAME supportive suite for storing, retrieving, and analyzing multi color microarray data. The system comprises a laboratory information management system (LIMS), a quality control management, as well as a sophisticated user management system. MARS is fully integrated into an analytical pipeline of microarray image analysis, normalization, gene expression clustering, and mapping of gene expression data onto biological pathways. The incorporation of ontologies and the use of MAGE-ML enables an export of studies stored in MARS to public repositories and other databases accepting these documents. Conclusion We have developed an integrated system tailored to serve the specific needs of microarray based research projects using a unique fusion of Web based and standalone applications connected to the latest J2EE application server technology. The presented system is freely available for academic and non-profit institutions. More information can be found at . PMID:15836795

  2. Profiling human serum antibodies with a carbohydrate antigen microarray

    PubMed Central

    Oyelaran, Oyindasola; McShane, Lisa M.; Dodd, Lori; Gildersleeve, Jeffrey C.

    2009-01-01

    Carbohydrate antigen arrays (glycan arrays) have been recently developed for the high-throughput analysis of carbohydrate macromolecule interactions. When profiling serum, information about experimental variability, inter-individual biological variability, and intra-individual temporal variability is critical. In this report, we describe the characterization of a carbohydrate antigen array and assay for profiling human serum. Through optimization of assay conditions and development of a normalization strategy, we obtain highly reproducible results with a within-experiment coefficient of variation (CV) of 10.8% and an overall CV (across multiple batches of slides and days) of 28.5%. We also report antibody profiles for 48 human subjects and evaluate for the first time the effects of age, race, sex, geographic location, and blood type on antibody profiles for a large set of carbohydrate antigens. We found significant dependence on age and blood type of antibody levels for a variety of carbohydrates. Finally, we conducted a longitudinal study with a separate group of 7 serum donors to evaluate the variation in anti-carbohydrate antibody levels within an individual over a period ranging from 3 to 13 weeks and found that, for nearly all antigens on our array, antibody levels are generally stable over this period. The results presented here provide the most comprehensive evaluation of experimental and biological variation reported to date for a glycan array and have significant implications for studies involving human serum profiling. PMID:19624168

  3. Lensfree holographic imaging of antibody microarrays for high-throughput detection of leukocyte numbers and function.

    PubMed

    Stybayeva, Gulnaz; Mudanyali, Onur; Seo, Sungkyu; Silangcruz, Jaime; Macal, Monica; Ramanculov, Erlan; Dandekar, Satya; Erlinger, Anthony; Ozcan, Aydogan; Revzin, Alexander

    2010-05-01

    Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.

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

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-06

    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.

  5. Microarray analysis in pulmonary hypertension

    PubMed Central

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea

    2016-01-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance. PMID:27076594

  6. Microarray analysis in pulmonary hypertension.

    PubMed

    Hoffmann, Julia; Wilhelm, Jochen; Olschewski, Andrea; Kwapiszewska, Grazyna

    2016-07-01

    Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance.

  7. The Impact of Photobleaching on Microarray Analysis

    PubMed Central

    von der Haar, Marcel; Preuß, John-Alexander; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2015-01-01

    DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results. PMID:26378589

  8. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

    PubMed Central

    Stieber, Bettina; Monecke, Stefan; Müller, Elke; Büchler, Joseph; Ehricht, Ralf

    2015-01-01

    Background S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins. Methods In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays. Results 110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate. Conclusions The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers. PMID:26624622

  9. MICROARRAY DATA ANALYSIS USING MULTIPLE STATISTICAL MODELS

    EPA Science Inventory

    Microarray Data Analysis Using Multiple Statistical Models

    Wenjun Bao1, Judith E. Schmid1, Amber K. Goetz1, Ming Ouyang2, William J. Welsh2,Andrew I. Brooks3,4, ChiYi Chu3,Mitsunori Ogihara3,4, Yinhe Cheng5, David J. Dix1. 1National Health and Environmental Effects Researc...

  10. Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays

    PubMed Central

    Gerdtsson, Anna S.; Dexlin-Mellby, Linda; Delfani, Payam; Berglund, Erica; Borrebaeck, Carl A. K.; Wingren, Christer

    2016-01-01

    Antibody microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of antibody microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant antibody microarray design. The results clearly demonstrated the importance of the surface-antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts. PMID:27600082

  11. Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays.

    PubMed

    Gerdtsson, Anna S; Dexlin-Mellby, Linda; Delfani, Payam; Berglund, Erica; Borrebaeck, Carl A K; Wingren, Christer

    2016-06-08

    Antibody microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of antibody microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant antibody microarray design. The results clearly demonstrated the importance of the surface-antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts.

  12. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  13. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  14. Technical Advances of the Recombinant Antibody Microarray Technology Platform for Clinical Immunoproteomics

    PubMed Central

    Delfani, Payam; Dexlin Mellby, Linda; Nordström, Malin; Holmér, Andreas; Ohlsson, Mattias; Borrebaeck, Carl A. K.; Wingren, Christer

    2016-01-01

    In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by antibody-based microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant antibody microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant antibody microarray technology platform designed for clinical immunoproteomics. PMID:27414037

  15. Integrated analysis of microarray data and gene function information.

    PubMed

    Cui, Yan; Zhou, Mi; Wong, Wing Hung

    2004-01-01

    Microarray data should be interpreted in the context of existing biological knowledge. Here we present integrated analysis of microarray data and gene function classification data using homogeneity analysis. Homogeneity analysis is a graphical multivariate statistical method for analyzing categorical data. It converts categorical data into graphical display. By simultaneously quantifying the microarray-derived gene groups and gene function categories, it captures the complex relations between biological information derived from microarray data and the existing knowledge about the gene function. Thus, homogeneity analysis provides a mathematical framework for integrating the analysis of microarray data and the existing biological knowledge.

  16. 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.

  17. 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

  18. Cluster of differentiation antibody microarrays on plasma immersion ion implanted polycarbonate.

    PubMed

    Kosobrodova, E; Mohamed, A; Su, Y; Kondyurin, A; dos Remedios, C G; McKenzie, D R; Bilek, M M M

    2014-02-01

    Plasma immersion ion implantation (PIII) modifies the surface properties of polymers, enabling them to covalently immobilize proteins without using linker chemistry. We describe the use of PIII treated polycarbonate (PC) slides as a novel platform for producing microarrays of cluster of differentiation (CD) antibodies. We compare their performance to identical antibody microarrays printed on nitrocellulose-coated glass slides that are currently the industry standard. Populations of leukocytes are applied to the CD microarrays and unbound cells are removed revealing patterns of differentially immobilized cells that are detected in a simple label-free approach by scanning the slides with visible light. Intra-slide and inter-slide reproducibility, densities of bound cells, and limits of detection were determined. Compared to the nitrocellulose-coated glass slides, PIII treated PC slides have a lower background noise, better sensitivity, and comparable or better reproducibility. They require three-fold lower antibody concentrations to yield equivalent signal strength, resulting in significant reductions in production cost. The improved transparency of PIII treated PC in the near-UV and visible wavelengths combined with superior immobilization of biomolecules makes them an attractive platform for a wide range of microarray applications.

  19. Anti-CD antibody microarray for human leukocyte morphology examination allows analyzing rare cell populations and suggesting preliminary diagnosis in leukemia.

    PubMed

    Khvastunova, Alina N; Kuznetsova, Sofya A; Al-Radi, Liubov S; Vylegzhanina, Alexandra V; Zakirova, Anna O; Fedyanina, Olga S; Filatov, Alexander V; Vorobjev, Ivan A; Ataullakhanov, Fazly

    2015-07-27

    We describe a method for leukocyte sorting by a microarray of anti-cluster-of-differentiation (anti-CD) antibodies and for preparation of the bound cells for morphological or cytochemical examination. The procedure results in a "sorted" smear with cells positive for certain surface antigens localised in predefined areas. The morphology and cytochemistry of the microarray-captured normal and neoplastic peripheral blood mononuclear cells are identical to the same characteristics in a smear. The microarray permits to determine the proportions of cells positive for the CD antigens on the microarray panel with high correlation with flow cytometry. Using the anti-CD microarray we show that normal granular lymphocytes and lymphocytes with radial segmentation of the nuclei are positive for CD3, CD8, CD16 or CD56 but not for CD4 or CD19. We also show that the described technique permits to obtain a pure leukemic cell population or to separate two leukemic cell populations on different antibody spots and to study their morphology or cytochemistry directly on the microarray. In cases of leukemias/lymphomas when circulating neoplastic cells are morphologically distinct, preliminary diagnosis can be suggested from full analysis of cell morphology, cytochemistry and their binding pattern on the microarray.

  20. Monoclonal antibody selection for interleukin-4 quantification using suspension arrays and forward-phase protein microarrays.

    PubMed

    Wang, L; Cole, K D; Peterson, A; He, Hua-Jun; Gaigalas, A K; Zong, Y

    2007-12-01

    A recombinant mouse interleukin-4 (IL-4) and three different purified rat antimouse IL-4 monoclonal antibodies (Mab) with different clonalities were employed as a model system. This system was used to examine monoclonal antibody effectiveness using both conventional and high-throughput measurement techniques to select antibodies for attaining the most sensitive detection of the recombinant IL-4 through the "sandwich-type" immunoassays. Surface plasmon resonance (SPR) measurements and two high-throughput methods, suspension arrays (also called multiplexed bead arrays) and forward-phase protein microarrays, predicted the same capture (BVD4-1D11) and detection (BVD6-24G2) antibody pair for the most sensitive detection of the recombinant cytokine. By using this antibody pair, we were able to detect as low as 2 pg/mL of IL-4 in buffer solution and 13.5 pg/mL of IL-4 spiked in 100% normal mouse serum with the multiplexed bead arrays. Due to the large amount of material required for SPR measurements, the study suggests that the multiplexed bead arrays and protein microarrays are both suited for the selection of numerous antibodies against the same analyte of interest to meet the need in the areas of systems biology and reproducible clinical diagnostics for better patient care.

  1. Meta-analysis of incomplete microarray studies.

    PubMed

    Zollinger, Alix; Davison, Anthony C; Goldstein, Darlene R

    2015-10-01

    Meta-analysis of microarray studies to produce an overall gene list is relatively straightforward when complete data are available. When some studies lack information-providing only a ranked list of genes, for example-it is common to reduce all studies to ranked lists prior to combining them. Since this entails a loss of information, we consider a hierarchical Bayes approach to meta-analysis using different types of information from different studies: the full data matrix, summary statistics, or ranks. The model uses an informative prior for the parameter of interest to aid the detection of differentially expressed genes. Simulations show that the new approach can give substantial power gains compared with classical meta-analysis and list aggregation methods. A meta-analysis of 11 published studies with different data types identifies genes known to be involved in ovarian cancer and shows significant enrichment.

  2. An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus.

    PubMed

    Kim, Sang-Woo; Kim, Min-Gon; Jung, Hyo-Am; Lee, Kyung-Hee; Lee, Hyun-Sook; Ro, Hyeon-Su

    2008-03-15

    The oyster mushroom spherical virus (OMSV) is a causative agent of dieback disease in the oyster mushroom, Pleurotus ostreatus. Outbreaks of this virus occasionally result in serious disease that is associated with hefty economic losses. Thus, the detection and removal of OMSV-infected spawn is considered to be a crucial step for the stable production of P. ostreatus. For the detection of OMSV, we attempted to generate monoclonal antibodies (mAbs) against an RNA polymerase domain (RPD) of an OMSV protein. In an effort to simplify the laborious multistep mAb screening process, we developed a protein microarray on a slide glass that is chemically modified with the RPD protein. The culture supernatants of 87 hybridoma cells, which were prepared from the fusion of RPD-immunized mouse spleen cells with myeloma cells, were spotted onto the RPD-coated microarray. The binding of mAb to RPD was detected via Alexa 488 dye-labeled anti-mouse immunoglobulin G (IgG) as a secondary antibody. Of 87 samples, 13 evidenced a significant level of fluorescence signal intensity. Subsequent immunoblot analysis revealed that the specificity of each mAb against RPD coincided with the corresponding fluorescence signal intensity, thereby indicating the effectiveness of the protein microarray in mAb screening.

  3. Software and tools for microarray data analysis.

    PubMed

    Mehta, Jai Prakash; Rani, Sweta

    2011-01-01

    A typical microarray experiment results in series of images, depending on the experimental design and number of samples. Software analyses the images to obtain the intensity at each spot and quantify the expression for each transcript. This is followed by normalization, and then various data analysis techniques are applied on the data. The whole analysis pipeline requires a large number of software to accurately handle the massive amount of data. Fortunately, there are large number of freely available and commercial software to churn the massive amount of data to manageable sets of differentially expressed genes, functions, and pathways. This chapter describes the software and tools which can be used to analyze the gene expression data right from the image analysis to gene list, ontology, and pathways.

  4. Evaluating mixtures of 14 hygroscopic additives to improve antibody microarray performance.

    PubMed

    Bergeron, Sébastien; Laforte, Veronique; Lo, Pik-Shan; Li, Huiyan; Juncker, David

    2015-11-01

    Microarrays allow the miniaturization and multiplexing of biological assays while only requiring minute amounts of samples. As a consequence of the small volumes used for spotting and the assays, evaporation often deteriorates the quality, reproducibility of spots, and the overall assay performance. Glycerol is commonly added to antibody microarray printing buffers to decrease evaporation; however, it often decreases the binding of antibodies to the surface, thereby negatively affecting assay sensitivity. Here, combinations of 14 hygroscopic chemicals were used as additives to printing buffers for contact-printed antibody microarrays on four different surface chemistries. The ability of the additives to suppress evaporation was quantified by measuring the residual buffer volume in open quill pins over time. The seven best additives were then printed either individually or as a 1:1 mixture of two additives, and the homogeneity, intensity, and reproducibility of both the spotted protein and of a fluorescently labeled analyte in an assay were quantified. Among the 28 combinations on the four slides, many were found to outperform glycerol, and the best additive mixtures were further evaluated by changing the ratio of the two additives. We observed that the optimal additive mixture was dependent on the slide chemistry, and that it was possible to increase the binding of antibodies to the surface threefold compared to 50 % glycerol, while decreasing whole-slide coefficient of variation to 5.9 %. For the two best slides, improvements were made for both the limit of detection (1.6× and 5.9×, respectively) and the quantification range (1.2× and 2.1×, respectively). The additive mixtures identified here thus help improve assay reproducibility and performance, and might be beneficial to all types of microarrays that suffer from evaporation of the printing buffers.

  5. Performance of a multiplexed serological microarray for the detection of antibodies against central nervous system pathogens.

    PubMed

    Jääskeläinen, Anne J; Viitala, Sari M; Kurkela, Satu; Hepojoki, Satu; Sillanpää, Heidi; Kallio-Kokko, Hannimari; Bergström, Tomas; Suni, Jukka; Närvänen, Ale; Vapalahti, Olli; Vaheri, Antti

    2014-05-01

    Central nervous system (CNS) infections have multiple potential causative agents for which simultaneous pathogen screening can provide a useful tool. This study evaluated a multiplexed microarray for the simultaneous detection of antibodies against CNS pathogens. The performance of selected microarray antigens for the detection of IgG antibodies against herpes simplex virus 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), adenovirus, Mycoplasma pneumoniae and Borrelia burgdorferi sensu lato, was evaluated using serum sample panels tested with reference assays used in a routine diagnostic laboratory. The microarray sensitivity for HSV-1, HSV-2, VZV, adenovirus and M. pneumonia ranged from 77% to 100%, and the specificity ranged from 74% to 97%. Very variable sensitivities and specificities were found for borrelial antigens of three different VlsE protein IR(6) peptide variants (IR6p1, IR6p2, IR6p4) and three recombinant decorin binding proteins A (DbpA; DbpAIa, DbpA91, DbpAG40). For single antigens, good specificity was shown for antigens of IR6p4 and DbpAIa (96%), while DbpA91, IR6p1 and IR6p2 were moderately specific (88-92%). The analytical sensitivity of the microarray was dependent on the borrelial IgG concentration of the specimen. The overall performance and technical features of the platform showed that the platform supports both recombinant proteins, whole viruses and peptides as antigens. This study showed diagnostic potential for all six CNS pathogens, including Borrelia burgdorferi sensu lato, using glutaraldehyde based microarray, and further highlighted the importance of careful antigen selection and the requirement for the use of multiple borrelial antigens in order to increase specificity without a major lack of sensitivity.

  6. Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays

    PubMed Central

    Sill, Martin; Schröder, Christoph; Shen, Ying; Marzoq, Aseel; Komel, Radovan; Hoheisel, Jörg D.; Nienhüser, Henrik; Schmidt, Thomas; Kastelic, Damjana

    2016-01-01

    In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL‐10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins’ capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches. PMID:27600085

  7. Genome-Scale Protein Microarray Comparison of Human Antibody Responses in Plasmodium vivax Relapse and Reinfection

    PubMed Central

    Chuquiyauri, Raul; Molina, Douglas M.; Moss, Eli L.; Wang, Ruobing; Gardner, Malcolm J.; Brouwer, Kimberly C.; Torres, Sonia; Gilman, Robert H.; Llanos-Cuentas, Alejandro; Neafsey, Daniel E.; Felgner, Philip; Liang, Xiaowu; Vinetz, Joseph M.

    2015-01-01

    Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions. PMID:26149860

  8. CYANOCHIP: an antibody microarray for high-taxonomical-resolution cyanobacterial monitoring.

    PubMed

    Blanco, Yolanda; Quesada, Antonio; Gallardo-Carreño, Ignacio; Aguirre, Jacobo; Parro, Victor

    2015-02-03

    Cyanobacteria are Gram-negative photosynthetic prokaryotes that are widespread on Earth. Eutrophication and global warming make some aquatic ecosystems behave as bioreactors that trigger rapid and massive cyanobacterial growth with remarkable economic and health consequences. Rapid and efficient early warning systems are required to support decisions by water body authorities. We have produced 17 specific antibodies to the most frequent cyanobacterial strains blooming in freshwater ecosystems, some of which are toxin producers. A sandwich-type antibody microarray immunoassay (CYANOCHIP) was developed for the simultaneous testing of any of the 17 strains, or other closely related strains, in field samples from different habitats (water, rocks, and sediments). We titrated and tested all of the antibodies in succession using a fluorescent sandwich microarray immunoassay. Although most showed high specificity, we applied a deconvolution method based on graph theory to disentangle the few existing cross-reactions. The CYANOCHIP sensitivity ranged from 10(2) to 10(4) cells mL(-1), with most antibodies detecting approximately 10(2) cells mL(-1). We validated the system by testing multiple isolates and crude natural samples from freshwater reservoirs and rocks, both in the laboratory and by in situ testing in the field. The results demonstrated that CYANOCHIP is a valuable tool for the sensitive and reliable detection of cyanobacteria for early warning and research purposes.

  9. Microarrays with Varying Carbohydrate Density Reveal Distinct Subpopulations of Serum Antibodies

    PubMed Central

    Oyelaran, Oyindasola; Li, Qian; Farnsworth, David; Gildersleeve, Jeffrey C.

    2009-01-01

    Antigen arrays have become important tools for profiling complex mixtures of proteins such as serum antibodies. These arrays can be used to better understand immune responses, discover new biomarkers, and guide the development of vaccines. Nevertheless, they are not perfect and improved array designs would enhance the information derived from this technology. In this study, we describe and evaluate a strategy for varying antigen density on an array and then use the array to study binding of lectins, monoclonal antibodies, and serum antibodies. To vary density, neoglycoproteins containing differing amounts of carbohydrate were synthesized and used to make a carbohydrate microarray with variations in both structure and density. We demonstrate that this method provides variations in density on the array surface within a range that is relevant for biological recognition events. The array was used to evaluate density dependent binding properties of three lectins (Vicia villosa lectin B4, Helix pomatia agglutinin, and soybean agglutinin) and three monoclonal antibodies (HBTn-1, B1.1, and Bric111) that bind the tumor-associated Tn antigen. In addition, serum antibodies were profiled from 30 healthy donors. The results show that variations in antigen density are required to detect the full spectrum of antibodies that bind a particular antigen and can be used to reveal differences in antibody populations between individuals that are not detectable using a single antigen density. PMID:19366269

  10. Tissue Microarray Analysis Applied to Bone Diagenesis

    PubMed Central

    Mello, Rafael Barrios; Silva, Maria Regina Regis; Alves, Maria Teresa Seixas; Evison, Martin Paul; Guimarães, Marco Aurelio; Francisco, Rafaella Arrabaca; Astolphi, Rafael Dias; Iwamura, Edna Sadayo Miazato

    2017-01-01

    Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered. PMID:28051148

  11. Transcriptome Analysis of Zebrafish Embryogenesis Using Microarrays

    PubMed Central

    Mathavan, Sinnakaruppan; Lee, Serene G. P; Mak, Alicia; Miller, Lance D; Murthy, Karuturi Radha Krishna; Govindarajan, Kunde R; Tong, Yan; Wu, Yi Lian; Lam, Siew Hong; Yang, Henry; Ruan, Yijun; Korzh, Vladimir; Gong, Zhiyuan; Liu, Edison T; Lufkin, Thomas

    2005-01-01

    Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html). PMID:16132083

  12. Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.

    PubMed

    Blanco, Yolanda; Moreno-Paz, Mercedes; Parro, Victor

    2017-02-07

    Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-μm filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 μm) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

  13. Polypyrrole-peptide microarray for biomolecular interaction analysis by SPR imaging

    PubMed Central

    Villiers, Marie-Bernadette; Cortès, Sandra; Brakha, Carine; Marche, Patrice; Roget, André; Livache, Thierry

    2009-01-01

    Nowadays, high-throughput analysis of biological events is a great challenge which could take benefit of the recent development of microarray devices. The great potential of such technology is related to the availability of a chip bearing a large set of probes, stable and easy to obtain, and suitable for ligand binding detection. Here, we described a new method based on polypyrrole chemistry and allowing the covalent immobilization of peptides in a microarray format and on a gold surface compatible with the use of Surface Plasmon Resonance. This technique is then illustrated by the detection and characterization of antibodies induced by hepatitis C virus and present in patients’serums. PMID:19649603

  14. Visualization of high-throughput and label-free antibody-polypeptide binding for drug screening based on microarrays and surface plasmon resonance imaging

    NASA Astrophysics Data System (ADS)

    Chen, Shengyi; Deng, Tao; Wang, Tongzhou; Wang, Jia; Li, Xin; Li, Qiang; Huang, Guoliang

    2012-01-01

    This work presents a visualization method for the high-throughput monitoring of antibody-polypeptide binding by integrating a microarray chip with surface plasmon resonance imaging (SPRi). A prism-coupled SPRi system with smart images processing software and a 5×5 polypeptide microarray was developed. The modeling analysis was performed to optimize the system and the materials of prism and chip, looking for the optimal incident wavelength and angle of incidence for dynamic SPRi detection in solution. The system can dynamically monitor 25 tunnels of biomolecule interactions in solution without secondary tag reactants. In addition, this system can determine the specific profile of antibody-polypeptide binding in each tunnel and yield a visual three-dimensional histogram of dynamic combinations in all microarray tunnels. Furthermore, the detection limit of the label-free antibody-polypeptide binding reached 1 pg/μL in a one-step binding test, and an ultrasensitive detection of 10 fg/μL was obtained using three-step cascade binding. Using the peptide microarray, the amount of sample and reagents used was reduced to 80 nL per tunnel, and 20×20 tunnels of biomolecule interactions could be analyzed in parallel in a 7 mm×7 mm microreaction cells. This device and method offer a potential platform for high-throughput and label-free dynamic monitoring multiple biomolecule interactions for drug discovery and basic biomedical research.

  15. Microarray screening of Guillain-Barré syndrome sera for antibodies to glycolipid complexes

    PubMed Central

    Halstead, Susan K.; Kalna, Gabriela; Islam, Mohammad B.; Jahan, Israt; Mohammad, Quazi D.; Jacobs, Bart C.; Endtz, Hubert P.; Islam, Zhahirul

    2016-01-01

    Objective: To characterize the patterns of autoantibodies to glycolipid complexes in a large cohort of Guillain-Barré syndrome (GBS) and control samples collected in Bangladesh using a newly developed microarray technique. Methods: Twelve commonly studied glycolipids and lipids, plus their 66 possible heteromeric complexes, totaling 78 antigens, were applied to polyvinylidene fluoride–coated slides using a microarray printer. Arrays were probed with 266 GBS and 579 control sera (2 μL per serum, diluted 1/50) and bound immunoglobulin G detected with secondary antibody. Scanned arrays were subjected to statistical analyses. Results: Measuring antibodies to single targets was 9% less sensitive than to heteromeric complex targets (49.2% vs 58.3%) without significantly affecting specificity (83.9%–85.0%). The optimal screening protocol for GBS sera comprised a panel of 10 glycolipids (4 single glycolipids GM1, GA1, GD1a, GQ1b, and their 6 heteromeric complexes), resulting in an overall assay sensitivity of 64.3% and specificity of 77.1%. Notable heteromeric targets were GM1:GD1a, GM1:GQ1b, and GA1:GD1a, in which exclusive binding to the complex was observed. Conclusions: Rationalizing the screening protocol to capture the enormous diversity of glycolipid complexes can be achieved by miniaturizing the screening platform to a microarray platform, and applying simple bioinformatics to determine optimal sensitivity and specificity of the targets. Glycolipid complexes are an important category of glycolipid antigens in autoimmune neuropathy cases that require specific analytical and bioinformatics methods for optimal detection. PMID:27790627

  16. A flow-through microarray cell for the online SERS detection of antibody-captured E. coli bacteria.

    PubMed

    Knauer, Maria; Ivleva, Natalia P; Niessner, Reinhard; Haisch, Christoph

    2012-03-01

    We present an immunoassay microarray flow-through system for the surface-enhanced Raman scattering (SERS) analysis of bacteria. The system has been constructed to support and automatize the nondestructive in situ analysis of different microorganisms in aqueous environment. After the immobilization of the desired antibodies to an activated PEG-coated surface, the chip is placed into the flow cell which is then flushed with the contaminated sample. Finally, colloidal metal nanoparticles are added and the cells are detected label-free by SERS. Here, we introduce the successful imaging of single microorganisms in the flow cell as well as the quantification of microorganisms in water by SERS mapping with a linear range between 4.3 × 10(3) to 4.3 × 10(5) cells/mL. The method has potential for routine application, e.g. for drinking water control.

  17. Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

    PubMed

    Pawlak, Michael; Schick, Eginhard; Bopp, Martin A; Schneider, Michael J; Oroszlan, Peter; Ehrat, Markus

    2002-04-01

    Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will

  18. Analysis of Microarray and RNA-seq Expression Profiling Data.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2017-03-01

    Gene expression profiling refers to the simultaneous measurement of the expression levels of a large number of genes (often all genes in a genome), typically in multiple experiments spanning a variety of cell types, treatments, or environmental conditions. Expression profiling is accomplished by assaying mRNA levels with microarrays or next-generation sequencing technologies (RNA-seq). This introduction describes normalization and analysis of data generated from microarray or RNA-seq experiments.

  19. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

    NASA Astrophysics Data System (ADS)

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N.; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

    2016-04-01

    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ˜100% sensitivity, ˜91% specificity and ˜96% accuracy. In the blinded test, the signals were classified with ˜91% sensitivity, ˜82% specificity and ˜86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ˜1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  20. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays.

    PubMed

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

    2016-04-01

    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

  1. Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

    PubMed Central

    Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

    2016-01-01

    We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ~100% sensitivity, ~91% specificity and ~96% accuracy. In the blinded test, the signals were classified with ~91% sensitivity, ~82% specificity and ~86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ~1—17 cells per 5 µl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays. PMID:26901310

  2. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    DOE PAGES

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; ...

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

  3. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    SciTech Connect

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

  4. ProMAT: protein microarray analysis tool

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Varnum, Susan M.; Anderson, Kevin K.; Bollinger, Nikki; Zangar, Richard C.

    2006-04-04

    Summary: ProMAT is a software tool for statistically analyzing data from ELISA microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality. The tool is available for Windows or Mac, and is distributed as open-source Java and R code. Availability: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions) which are distributed with the tool.

  5. WebArray: an online platform for microarray data analysis

    PubMed Central

    Xia, Xiaoqin; McClelland, Michael; Wang, Yipeng

    2005-01-01

    Background Many cutting-edge microarray analysis tools and algorithms, including commonly used limma and affy packages in Bioconductor, need sophisticated knowledge of mathematics, statistics and computer skills for implementation. Commercially available software can provide a user-friendly interface at considerable cost. To facilitate the use of these tools for microarray data analysis on an open platform we developed an online microarray data analysis platform, WebArray, for bench biologists to utilize these tools to explore data from single/dual color microarray experiments. Results The currently implemented functions were based on limma and affy package from Bioconductor, the spacings LOESS histogram (SPLOSH) method, PCA-assisted normalization method and genome mapping method. WebArray incorporates these packages and provides a user-friendly interface for accessing a wide range of key functions of limma and others, such as spot quality weight, background correction, graphical plotting, normalization, linear modeling, empirical bayes statistical analysis, false discovery rate (FDR) estimation, chromosomal mapping for genome comparison. Conclusion WebArray offers a convenient platform for bench biologists to access several cutting-edge microarray data analysis tools. The website is freely available at . It runs on a Linux server with Apache and MySQL. PMID:16371165

  6. Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM).

    PubMed

    Halloran, P F; Pereira, A B; Chang, J; Matas, A; Picton, M; De Freitas, D; Bromberg, J; Serón, D; Sellarés, J; Einecke, G; Reeve, J

    2013-11-01

    In a reference set of 403 kidney transplant biopsies, we recently developed a microarray-based test that diagnoses antibody-mediated rejection (ABMR) by assigning an ABMR score. To validate the ABMR score and assess its potential impact on practice, we performed the present prospective INTERCOM study (clinicaltrials.gov NCT01299168) in 300 new biopsies (264 patients) from six centers: Baltimore, Barcelona, Edmonton, Hannover, Manchester and Minneapolis. We assigned ABMR scores using the classifier created in the reference set and compared it to conventional assessment as documented in the pathology reports. INTERCOM documented uncertainty in conventional assessment: In 41% of biopsies where ABMR features were noted, the recorded diagnoses did not mention ABMR. The ABMR score correlated with ABMR histologic lesions and donor-specific antibodies, but not with T cell-mediated rejection lesions. The agreement between ABMR scores and conventional assessment was identical to that in the reference set (accuracy 85%). The ABMR score was more strongly associated with failure than conventional assessment, and when the ABMR score and conventional assessment disagreed, only the ABMR score was associated with early progression to failure. INTERCOM confirms the need to reduce uncertainty in the diagnosis of ABMR, and demonstrates the potential of the ABMR score to impact practice.

  7. Short time-series microarray analysis: Methods and challenges

    PubMed Central

    Wang, Xuewei; Wu, Ming; Li, Zheng; Chan, Christina

    2008-01-01

    The detection and analysis of steady-state gene expression has become routine. Time-series microarrays are of growing interest to systems biologists for deciphering the dynamic nature and complex regulation of biosystems. Most temporal microarray data only contain a limited number of time points, giving rise to short-time-series data, which imposes challenges for traditional methods of extracting meaningful information. To obtain useful information from the wealth of short-time series data requires addressing the problems that arise due to limited sampling. Current efforts have shown promise in improving the analysis of short time-series microarray data, although challenges remain. This commentary addresses recent advances in methods for short-time series analysis including simplification-based approaches and the integration of multi-source information. Nevertheless, further studies and development of computational methods are needed to provide practical solutions to fully exploit the potential of this data. PMID:18605994

  8. Comparative analysis of genomic signal processing for microarray data clustering.

    PubMed

    Istepanian, Robert S H; Sungoor, Ala; Nebel, Jean-Christophe

    2011-12-01

    Genomic signal processing is a new area of research that combines advanced digital signal processing methodologies for enhanced genetic data analysis. It has many promising applications in bioinformatics and next generation of healthcare systems, in particular, in the field of microarray data clustering. In this paper we present a comparative performance analysis of enhanced digital spectral analysis methods for robust clustering of gene expression across multiple microarray data samples. Three digital signal processing methods: linear predictive coding, wavelet decomposition, and fractal dimension are studied to provide a comparative evaluation of the clustering performance of these methods on several microarray datasets. The results of this study show that the fractal approach provides the best clustering accuracy compared to other digital signal processing and well known statistical methods.

  9. Optimized T7 amplification system for microarray analysis.

    PubMed

    Pabón, C; Modrusan, Z; Ruvolo, M V; Coleman, I M; Daniel, S; Yue, H; Arnold, L J

    2001-10-01

    Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

  10. Microarray analysis of DNA replication timing.

    PubMed

    Karnani, Neerja; Taylor, Christopher M; Dutta, Anindya

    2009-01-01

    Although all of the DNA in an eukaryotic cell replicates during the S-phase of cell cycle, there is a significant difference in the actual time in S-phase when a given chromosomal segment replicates. Methods are described here for generation of high-resolution temporal maps of DNA replication in synchronized human cells. This method does not require amplification of DNA before microarray hybridization and so avoids errors introduced during PCR. A major advantage of using this procedure is that it facilitates finer dissection of replication time in S-phase. Also, it helps delineate chromosomal regions that undergo biallelic or asynchronous replication, which otherwise are difficult to detect at a genome-wide scale by existing methods. The continuous TR50 (time of completion of 50% replication) maps of replication across chromosomal segments identify regions that undergo acute transitions in replication timing. These transition zones can play a significant role in identifying insulators that separate chromosomal domains with different chromatin modifications.

  11. Determination of specific antibody responses to the six species of ebola and Marburg viruses by multiplexed protein microarrays.

    PubMed

    Kamata, Teddy; Natesan, Mohan; Warfield, Kelly; Aman, M Javad; Ulrich, Robert G

    2014-12-01

    Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses.

  12. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  13. 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.

  14. SOLID3: A Multiplex Antibody Microarray-Based Optical Sensor Instrument for In Situ Life Detection in Planetary Exploration

    NASA Astrophysics Data System (ADS)

    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 1-2ppb (ngmL-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 50mM 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.

  15. 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.

  16. Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos)

    PubMed Central

    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. PMID:27293753

  17. Independent component analysis of Alzheimer's DNA microarray gene expression data

    PubMed Central

    Kong, Wei; Mou, Xiaoyang; Liu, Qingzhong; Chen, Zhongxue; Vanderburg, Charles R; Rogers, Jack T; Huang, Xudong

    2009-01-01

    Background Gene microarray technology is an effective tool to investigate the simultaneous activity of multiple cellular pathways from hundreds to thousands of genes. However, because data in the colossal amounts generated by DNA microarray technology are usually complex, noisy, high-dimensional, and often hindered by low statistical power, their exploitation is difficult. To overcome these problems, two kinds of unsupervised analysis methods for microarray data: principal component analysis (PCA) and independent component analysis (ICA) have been developed to accomplish the task. PCA projects the data into a new space spanned by the principal components that are mutually orthonormal to each other. The constraint of mutual orthogonality and second-order statistics technique within PCA algorithms, however, may not be applied to the biological systems studied. Extracting and characterizing the most informative features of the biological signals, however, require higher-order statistics. Results ICA is one of the unsupervised algorithms that can extract higher-order statistical structures from data and has been applied to DNA microarray gene expression data analysis. We performed FastICA method on DNA microarray gene expression data from Alzheimer's disease (AD) hippocampal tissue samples and consequential gene clustering. Experimental results showed that the ICA method can improve the clustering results of AD samples and identify significant genes. More than 50 significant genes with high expression levels in severe AD were extracted, representing immunity-related protein, metal-related protein, membrane protein, lipoprotein, neuropeptide, cytoskeleton protein, cellular binding protein, and ribosomal protein. Within the aforementioned categories, our method also found 37 significant genes with low expression levels. Moreover, it is worth noting that some oncogenes and phosphorylation-related proteins are expressed in low levels. In comparison to the PCA and support

  18. Microarray data analysis for differential expression: a tutorial.

    PubMed

    Suárez, Erick; Burguete, Ana; Mclachlan, Geoffrey J

    2009-06-01

    DNA microarray is a technology that simultaneously evaluates quantitative measurements for the expression of thousands of genes. DNA microarrays have been used to assess gene expression between groups of cells of different organs or different populations. In order to understand the role and function of the genes, one needs the complete information about their mRNA transcripts and proteins. Unfortunately, exploring the protein functions is very difficult, due to their unique 3-dimentional complicated structure. To overcome this difficulty, one may concentrate on the mRNA molecules produced by the gene expression. In this paper, we describe some of the methods for preprocessing data for gene expression and for pairwise comparison from genomic experiments. Previous studies to assess the efficiency of different methods for pairwise comparisons have found little agreement in the lists of significant genes. Finally, we describe the procedures to control false discovery rates, sample size approach for these experiments, and available software for microarray data analysis. This paper is written for those professionals who are new in microarray data analysis for differential expression and want to have an overview of the specific steps or the different approaches for this sort of analysis.

  19. Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection

    DTIC Science & Technology

    2010-03-19

    sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of...Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection John...Sensitive Immunoassay Using Electrochemical Detection 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT

  20. Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform.

    PubMed

    Landry, J P; Ke, Yaohuang; Yu, Guo-Liang; Zhu, X D

    2015-02-01

    Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

  1. Deciphering the Prokaryotic Community and Metabolisms in South African Deep-Mine Biofilms through Antibody Microarrays and Graph Theory

    PubMed Central

    García-Moyano, Antonio; Aguirre, Jacobo; Cruz-Gil, Patricia; Palacín, Arantxa; van Heerden, Esta; Parro, Víctor

    2014-01-01

    In the South African deep mines, a variety of biofilms growing in mine corridor walls as water seeps from intersections or from fractures represents excellent proxies for deep-subsurface environments. However, they may be greatly affected by the oxygen inputs through the galleries of mining activities. As a consequence, the interaction between the anaerobic water coming out from the walls with the oxygen inputs creates new conditions that support rich microbial communities. The inherent difficulties for sampling these delicate habitats, together with transport and storage conditions may alter the community features and composition. Therefore, the development of in situ monitoring methods would be desirable for quick evaluation of the microbial community. In this work, we report the usefulness of an antibody-microarray (EMChip66) immunoassay for a quick check of the microbial diversity of biofilms located at 1.3 km below surface within the Beatrix deep gold mine (South Africa). In addition, a deconvolution method, previously described and used for environmental monitoring, based on graph theory and applied on antibody cross-reactivity was used to interpret the immunoassay results. The results were corroborated and further expanded by 16S rRNA gene sequencing analysis. Both culture-independent techniques coincided in detecting features related to aerobic sulfur-oxidizers, aerobic chemoorganotrophic Alphaproteobacteria and metanotrophic Gammaproteobacteria. 16S rRNA gene sequencing detected phylotypes related to nitrate-reducers and anaerobic sulfur-oxidizers, whereas the EMChip66 detected immunological features from methanogens and sulfate-reducers. The results reveal a diverse microbial community with syntrophic metabolisms both anaerobic (fermentation, methanogenesis, sulphate and nitrate reduction) and aerobic (methanotrophy, sulphur oxidation). The presence of oxygen-scavenging microbes might indicate that the system is modified by the artificial oxygen inputs

  2. Bioinformatics and Microarray Data Analysis on the Cloud.

    PubMed

    Calabrese, Barbara; Cannataro, Mario

    2016-01-01

    High-throughput platforms such as microarray, mass spectrometry, and next-generation sequencing are producing an increasing volume of omics data that needs large data storage and computing power. Cloud computing offers massive scalable computing and storage, data sharing, on-demand anytime and anywhere access to resources and applications, and thus, it may represent the key technology for facing those issues. In fact, in the recent years it has been adopted for the deployment of different bioinformatics solutions and services both in academia and in the industry. Although this, cloud computing presents several issues regarding the security and privacy of data, that are particularly important when analyzing patients data, such as in personalized medicine. This chapter reviews main academic and industrial cloud-based bioinformatics solutions; with a special focus on microarray data analysis solutions and underlines main issues and problems related to the use of such platforms for the storage and analysis of patients data.

  3. Microarray analysis of gene expression in medicinal plant research.

    PubMed

    Youns, M; Efferth, T; Hoheisel, J D

    2009-10-01

    Expression profiling analysis offers great opportunities for the identification of novel molecular targets, drug discovery, development, and validation. The beauty of microarray analysis of gene expression is that it can be used to screen the expression of tens of thousands of genes in parallel and to identify appropriate molecular targets for therapeutic intervention. Toward identifying novel therapeutic options, natural products, notably from medicinal plants used in traditional Chinese medicine (TCM), have been thoroughly investigated. Increased knowledge of the molecular mechanisms of TCM-derived drugs could be achieved through application of modern molecular technologies including transcript profiling. In the present review, we introduce a brief introduction to the field of microarray technology and disclose its role in target identification and validation. Moreover, we provide examples for applications regarding molecular target discovery in medicinal plants derived TCM. This could be an attractive strategy for the development of novel and improved therapeutics.

  4. MAGMA: analysis of two-channel microarrays made easy.

    PubMed

    Rehrauer, Hubert; Zoller, Stefan; Schlapbach, Ralph

    2007-07-01

    The web application MAGMA provides a simple and intuitive interface to identify differentially expressed genes from two-channel microarray data. While the underlying algorithms are not superior to those of similar web applications, MAGMA is particularly user friendly and can be used without prior training. The user interface guides the novice user through the most typical microarray analysis workflow consisting of data upload, annotation, normalization and statistical analysis. It automatically generates R-scripts that document MAGMA's entire data processing steps, thereby allowing the user to regenerate all results in his local R installation. The implementation of MAGMA follows the model-view-controller design pattern that strictly separates the R-based statistical data processing, the web-representation and the application logic. This modular design makes the application flexible and easily extendible by experts in one of the fields: statistical microarray analysis, web design or software development. State-of-the-art Java Server Faces technology was used to generate the web interface and to perform user input processing. MAGMA's object-oriented modular framework makes it easily extendible and applicable to other fields and demonstrates that modern Java technology is also suitable for rather small and concise academic projects. MAGMA is freely available at www.magma-fgcz.uzh.ch.

  5. Time-Frequency Analysis of Peptide Microarray Data: Application to Brain Cancer Immunosignatures

    PubMed Central

    O’Donnell, Brian; Maurer, Alexander; Papandreou-Suppappola, Antonia; Stafford, Phillip

    2015-01-01

    One of the gravest dangers facing cancer patients is an extended symptom-free lull between tumor initiation and the first diagnosis. Detection of tumors is critical for effective intervention. Using the body’s immune system to detect and amplify tumor-specific signals may enable detection of cancer using an inexpensive immunoassay. Immunosignatures are one such assay: they provide a map of antibody interactions with random-sequence peptides. They enable detection of disease-specific patterns using classic train/test methods. However, to date, very little effort has gone into extracting information from the sequence of peptides that interact with disease-specific antibodies. Because it is difficult to represent all possible antigen peptides in a microarray format, we chose to synthesize only 330,000 peptides on a single immunosignature microarray. The 330,000 random-sequence peptides on the microarray represent 83% of all tetramers and 27% of all pentamers, creating an unbiased but substantial gap in the coverage of total sequence space. We therefore chose to examine many relatively short motifs from these random-sequence peptides. Time-variant analysis of recurrent subsequences provided a means to dissect amino acid sequences from the peptides while simultaneously retaining the antibody–peptide binding intensities. We first used a simple experiment in which monoclonal antibodies with known linear epitopes were exposed to these random-sequence peptides, and their binding intensities were used to create our algorithm. We then demonstrated the performance of the proposed algorithm by examining immunosignatures from patients with Glioblastoma multiformae (GBM), an aggressive form of brain cancer. Eight different frameshift targets were identified from the random-sequence peptides using this technique. If immune-reactive antigens can be identified using a relatively simple immune assay, it might enable a diagnostic test with sufficient sensitivity to detect tumors

  6. Analysis of variance of microarray data.

    PubMed

    Ayroles, Julien F; Gibson, Greg

    2006-01-01

    Analysis of variance (ANOVA) is an approach used to identify differentially expressed genes in complex experimental designs. It is based on testing for the significance of the magnitude of effect of two or more treatments taking into account the variance within and between treatment classes. ANOVA is a highly flexible analytical approach that allows investigators to simultaneously assess the contributions of multiple factors to gene expression variation, including technical (dye, batch) effects and biological (sex, genotype, drug, time) ones, as well as interactions between factors. This chapter provides an overview of the theory of linear mixture modeling and the sequence of steps involved in fitting gene-specific models and discusses essential features of experimental design. Commercial and open-source software for performing ANOVA is widely available.

  7. Identification of candidate genes in osteoporosis by integrated microarray analysis

    PubMed Central

    Li, J. J.; Wang, B. Q.; Yang, Y.; Li, D.

    2016-01-01

    Objectives In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. Methods We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and

  8. Automated target preparation for microarray-based gene expression analysis.

    PubMed

    Raymond, Frédéric; Metairon, Sylviane; Borner, Roland; Hofmann, Markus; Kussmann, Martin

    2006-09-15

    DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.

  9. Microarray analysis of gene expression profiles in ripening pineapple fruits

    PubMed Central

    2012-01-01

    Background Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the

  10. Signal enhancement in antibody microarrays using quantum dots nanocrystals: application to potential Alzheimer's disease biomarker screening.

    PubMed

    Morales-Narváez, Eden; Montón, Helena; Fomicheva, Anna; Merkoçi, Arben

    2012-08-07

    The performance of cadmium-selenide/zinc-sulfide (CdSe@ZnS) quantum dots (QDs) and the fluorescent dye Alexa 647 as reporter in an assay designed to detect apolipoprotein E (ApoE) has been compared. The assay is a sandwich immunocomplex microarray that functions via excitation by visible light. ApoE was chosen for its potential as a biomarker for Alzheimer's disease. The two versions of the microarray (QD or Alexa 647) were assessed under the same experimental conditions and then compared to a conventional enzyme-linked immunosorbent assay (ELISA) targeting ApoE. The QDs proved to be highly effective reporters in the microarrays, although their performance strongly varied in function of the excitation wavelength. At 633 nm, the QD microarray gave a limit of detection (LOD) of ~247 pg mL(-1); however, at an excitation wavelength of 532 nm, it provided a LOD of ~62 pg mL(-1), five times more sensitive than that of the Alexa microarray (~307 pg mL(-1)) and seven times more than that of the ELISA (~470 pg mL(-1)). Finally, serial dilutions from a human serum sample were assayed with high sensitivity and acceptable precision and accuracy.

  11. Diagnostic challenges for multiplexed protein microarrays.

    PubMed

    Master, Stephen R; Bierl, Charlene; Kricka, Larry J

    2006-11-01

    Multiplexed protein analysis using planar microarrays or microbeads is growing in popularity for simultaneous assays of antibodies, cytokines, allergens, drugs and hormones. However, this new assay format presents several new operational issues for the clinical laboratory, such as the quality control of protein-microarray-based assays, the release of unrequested test data and the use of diagnostic algorithms to transform microarray data into diagnostic results.

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

    DTIC Science & Technology

    2006-04-27

    SRM117, 1026b, and 576 (micromoles of rhamnose equivalents per milliliter) were 3.6, 16.0, 3.6, and 3.5, respectively. We also used inulin (Sigma...the rabbit antiserum did not react with inulin , the polysaccharide used as a negative control. Furthermore, using this microarray, we are able to

  13. DNA Microarray Data Analysis: A Novel Biclustering Algorithm Approach

    NASA Astrophysics Data System (ADS)

    Tchagang, Alain B.; Tewfik, Ahmed H.

    2006-12-01

    Biclustering algorithms refer to a distinct class of clustering algorithms that perform simultaneous row-column clustering. Biclustering problems arise in DNA microarray data analysis, collaborative filtering, market research, information retrieval, text mining, electoral trends, exchange analysis, and so forth. When dealing with DNA microarray experimental data for example, the goal of biclustering algorithms is to find submatrices, that is, subgroups of genes and subgroups of conditions, where the genes exhibit highly correlated activities for every condition. In this study, we develop novel biclustering algorithms using basic linear algebra and arithmetic tools. The proposed biclustering algorithms can be used to search for all biclusters with constant values, biclusters with constant values on rows, biclusters with constant values on columns, and biclusters with coherent values from a set of data in a timely manner and without solving any optimization problem. We also show how one of the proposed biclustering algorithms can be adapted to identify biclusters with coherent evolution. The algorithms developed in this study discover all valid biclusters of each type, while almost all previous biclustering approaches will miss some.

  14. Using antibodies against ATPase and microarray immunoassays for the search for potential extraterrestrial life in saline environments on Mars.

    NASA Astrophysics Data System (ADS)

    Weigl, Andreas; Gruber, Claudia; Blanco-López, Yolanda; Rivas, Luis A.; Parro, Victor; Stan-Lotter, Helga

    2010-05-01

    membrane fraction and whole cell preparation of Halobacterium salinarum NRC-1, Escherichia coli LE392 as well as the whole cell fraction of Halorubrum saccharovorum and Bacillus megaterium. Further experiments with antibodies against ATPase are proposed to be done with procedures that are more adjusted to the search for extraterrestrial life. Therefore tests with a microarray system (Rivas et al., 2008) were done at the Centro de Astrobiología in Madrid. Cellular extracts of environmental samples from a sea salt from Piranske (Slovenia) and a rock salt from Himalaya (Pakistan) were tested with a "supermix" of 300 antibodies, additionally including an antibody against the subunit A of the A-ATPase from Halorubrum sacharovorum. Positive immuno reactions with antibodies against halophile cells as well as antibodies against exopolysaccharides could be shown. (1)Gruber C, Stan-Lotter H (1997) Western blot of stained proteins from dried polyacrylamide gels. Anal Biochem 253, 125-127. (2)Rivas LA, Garcia-Villadangos M, Moreno-Paz M, Cruz-Gil P, Gómez-Elvira J, Parro V (2008) A 200-antibody microarray biochip for environmental monitoring: searching for universal microbial biomarkers through immunoprofiling. Anal Chem 80, 7970-7979

  15. Comparison of microarray and sage techniques in gene expression analysis of human glioblastoma.

    PubMed

    Kavsan, V M; Dmitrenko, V V; Shostak, K O; Bukreieva, T V; Vitak, N Y; Simirenko, O E; Malisheva, T A; Shamayev, M I; Rozumenko, V D; Zozulya, Y A

    2007-01-01

    To enhance glioblastoma (GB) marker discovery we compared gene expression in GB with human normal brain (NB) by accessing SAGE Genie web site and compared obtained results with published data. Nine GB and five NB SAGE-libraries were analyzed using the Digital Gene Expression Displayer (DGED), the results of DGED were tested by Northern blot analysis and RT-PCR of arbitrary selected genes. Review of available data from the articles on gene expression profiling by microarray-based hybridization showed as few as 35 overlapped genes with increased expression in GB. Some of them were identified in four articles, but most genes in three or even in two investigations. There was found also some differences between SAGE results of GB analysis. Digital Gene Expression Displayer approach revealed 676 genes differentially expressed in GB vs. NB with cut-off ratio: twofold change and P < or = 0.05. Differential expression of selectedgenes obtained by DGED was confirmed by Northern analysis and RT-PCR. Altogether, only 105 of 955 genes presented in published investigations were among the genes obtained by DGED. Comparison of the results obtained by microarrays and SAGE is very complicated because authors present only the most prominent differentially expressed genes. However, even available data give quite poor overlapping of genes revealed by microarrays. Some differences between results obtained by SAGE in different investigations can be explained by high dependence on the statistical methods used. As for now, the best solution to search for molecular tumor markers is to compare all available results and to select only those genes, which significant expression in tumor combined with very low expression in normal tissues was reproduced in several articles. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of GBs. Some genes, encoded cell surface or extra-cellular proteins may be useful for targeting

  16. Structural analysis of hepatitis C RNA genome using DNA microarrays

    PubMed Central

    Martell, María; Briones, Carlos; de Vicente, Aránzazu; Piron, María; Esteban, Juan I.; Esteban, Rafael; Guardia, Jaime; Gómez, Jordi

    2004-01-01

    Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5′ non-coding region (5′NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions. PMID:15247323

  17. Portable system for microbial sample preparation and oligonucleotide microarray analysis.

    SciTech Connect

    Bavykin, S. G.; Akowski, J. P.; Zakhariev, V. M.; Barsky, V. E.; Mirzabekov, A. D.; Perov, A. N.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2001-02-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.

  18. Exon Microarray Analysis of Human Dorsolateral Prefrontal Cortex in Alcoholism

    PubMed Central

    Manzardo, Ann M.; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G.

    2014-01-01

    Background Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Methods Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC, Brodmann area 9) of 7 adult Alcoholic (6 males, 1 female, mean age 48 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST Array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using qRT-PCR, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Results Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN) and signaling (e.g., RASGRP, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease, and development including cellular assembly and organization impacting on psychological disorders. Conclusions Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation and signaling that targets white matter of the brain. PMID:24890784

  19. Using Kepler for Tool Integration in Microarray Analysis Workflows.

    PubMed

    Gan, Zhuohui; Stowe, Jennifer C; Altintas, Ilkay; McCulloch, Andrew D; Zambon, Alexander C

    Increasing numbers of genomic technologies are leading to massive amounts of genomic data, all of which requires complex analysis. More and more bioinformatics analysis tools are being developed by scientist to simplify these analyses. However, different pipelines have been developed using different software environments. This makes integrations of these diverse bioinformatics tools difficult. Kepler provides an open source environment to integrate these disparate packages. Using Kepler, we integrated several external tools including Bioconductor packages, AltAnalyze, a python-based open source tool, and R-based comparison tool to build an automated workflow to meta-analyze both online and local microarray data. The automated workflow connects the integrated tools seamlessly, delivers data flow between the tools smoothly, and hence improves efficiency and accuracy of complex data analyses. Our workflow exemplifies the usage of Kepler as a scientific workflow platform for bioinformatics pipelines.

  20. Analysis of Mycobacterium leprae gene expression using DNA microarray.

    PubMed

    Akama, Takeshi; Tanigawa, Kazunari; Kawashima, Akira; Wu, Huhehasi; Ishii, Norihisa; Suzuki, Koichi

    2010-10-01

    Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

  1. 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.

  2. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

    PubMed Central

    von der Haar, Marcel; Heuer, Christopher; Pähler, Martin; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2016-01-01

    The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs. PMID:27916881

  3. Cross-platform analysis of cancer microarray data improves gene expression based classification of phenotypes

    PubMed Central

    Warnat, Patrick; Eils, Roland; Brors, Benedikt

    2005-01-01

    Background The extensive use of DNA microarray technology in the characterization of the cell transcriptome is leading to an ever increasing amount of microarray data from cancer studies. Although similar questions for the same type of cancer are addressed in these different studies, a comparative analysis of their results is hampered by the use of heterogeneous microarray platforms and analysis methods. Results In contrast to a meta-analysis approach where results of different studies are combined on an interpretative level, we investigate here how to directly integrate raw microarray data from different studies for the purpose of supervised classification analysis. We use median rank scores and quantile discretization to derive numerically comparable measures of gene expression from different platforms. These transformed data are then used for training of classifiers based on support vector machines. We apply this approach to six publicly available cancer microarray gene expression data sets, which consist of three pairs of studies, each examining the same type of cancer, i.e. breast cancer, prostate cancer or acute myeloid leukemia. For each pair, one study was performed by means of cDNA microarrays and the other by means of oligonucleotide microarrays. In each pair, high classification accuracies (> 85%) were achieved with training and testing on data instances randomly chosen from both data sets in a cross-validation analysis. To exemplify the potential of this cross-platform classification analysis, we use two leukemia microarray data sets to show that important genes with regard to the biology of leukemia are selected in an integrated analysis, which are missed in either single-set analysis. Conclusion Cross-platform classification of multiple cancer microarray data sets yields discriminative gene expression signatures that are found and validated on a large number of microarray samples, generated by different laboratories and microarray technologies

  4. The application of phenotypic microarray analysis to anti-fungal drug development.

    PubMed

    Greetham, Darren; Lappin, David F; Rajendran, Ranjith; O'Donnell, Lindsay; Sherry, Leighann; Ramage, Gordon; Nile, Christopher

    2017-03-01

    Candida albicans metabolic activity in the presence and absence of acetylcholine was measured using phenotypic microarray analysis. Acetylcholine inhibited C. albicans biofilm formation by slowing metabolism independent of biofilm forming capabilities. Phenotypic microarray analysis can therefore be used for screening compound libraries for novel anti-fungal drugs and measuring antifungal resistance.

  5. Experimental Approaches to Microarray Analysis of Tumor Samples

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

    2008-01-01

    Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has…

  6. On the Statics for Micro-Array Data Analysis

    NASA Astrophysics Data System (ADS)

    Urushibara, Tomoko; Akasaka, Shizu; Ito, Makiko; Suzuki, Tomonori; Miyazaki, Satoru

    2010-01-01

    data, we might get a different result because the distinct definition for micro array data has not been set yet. It means that from the same data we will get different results depending on researchers. We are afraid that this problem will have a big effect on developing new medicines and to progress the next step, like a 2nd screening. So, we suggest that we should have certain guidelines to analyze Micro-Array data validly with statistic method and it will surely be helpful for Micro-Array analysis for medical studies in the future.

  7. Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach

    PubMed Central

    Kohler, Christian; Dunachie, Susanna J.; Müller, Elke; Kohler, Anne; Jenjaroen, Kemajittra; Teparrukkul, Prapit; Baier, Vico; Ehricht, Ralf; Steinmetz, Ivo

    2016-01-01

    Background The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking. Methods and Principal Findings In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response. Conclusions Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our

  8. Outcome Based Screening for Prognostic Phospho-RTK (Receptor Tyrosine Kinase) Antibodies Using Tissue Microarrays)

    DTIC Science & Technology

    2004-08-01

    T., Maffe, A., Maggiora, P., Stefani, A. D., from human breast cancer cells. Monoclonal antibody production isolation, and Valente , G., Giordano , S...Zhen, Z_. Giordano , S.. Longati, P., Medico. E., Campiglio. M., and Comoglio, P. M. 35. Mars. W. M., Zamegar. R., and Michalopoulos, G. K. Activation

  9. An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Intoxication and infection caused by foodborne pathogens are important problems in the United States, and screening tests for multiple pathogen detection have been developed because food producers are known reservoirs of multiple pathogens. We developed a 96-well microplate, multiplex antibody micr...

  10. Microarray Technology for Major Chemical Contaminants Analysis in Food: Current Status and Prospects

    PubMed Central

    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. PMID:23012541

  11. Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection

    PubMed Central

    Hospach, Ingeborg; Joseph, Yvonne; Mai, Michaela Kathrin; Krasteva, Nadejda; Nelles, Gabriele

    2014-01-01

    Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established. PMID:27600349

  12. A comparative analysis of DNA barcode microarray feature size

    PubMed Central

    Ammar, Ron; Smith, Andrew M; Heisler, Lawrence E; Giaever, Guri; Nislow, Corey

    2009-01-01

    Background Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity. Results We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO) collection used for screens of pooled yeast (Saccharomyces cerevisiae) deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 μm features sizes independently identified the primary target of tunicamycin to be ALG7. Conclusion We show that the data obtained with 5 μm feature size is of comparable quality to the 30 μm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density. PMID:19825181

  13. Design and analysis of mismatch probes for long oligonucleotide microarrays

    SciTech Connect

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  14. ArrayNinja: An Open Source Platform for Unified Planning and Analysis of Microarray Experiments

    PubMed Central

    Dickson, B.M.; Cornett, E.M.; Ramjan, Z.; Rothbart, S.B.

    2017-01-01

    Microarray-based proteomic platforms have emerged as valuable tools for studying various aspects of protein function, particularly in the field of chromatin biochemistry. Microarray technology itself is largely unrestricted in regard to printable material and platform design, and efficient multidimensional optimization of assay parameters requires fluidity in the design and analysis of custom print layouts. This motivates the need for streamlined software infrastructure that facilitates the combined planning and analysis of custom microarray experiments. To this end, we have developed ArrayNinja as a portable, open source, and interactive application that unifies the planning and visualization of microarray experiments and provides maximum flexibility to end users. Array experiments can be planned, stored to a private database, and merged with the imaged results for a level of data interaction and centralization that is not currently attainable with available microarray informatics tools. PMID:27423857

  15. Microarray analysis of Xenopus endoderm expressing Ptf1a

    PubMed Central

    Bilogan, Cassandra K.; Horb, Marko E.

    2012-01-01

    Pancreas specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this paper, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification. PMID:22815262

  16. A genome-wide 20 K citrus microarray for gene expression analysis

    PubMed Central

    Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

    2008-01-01

    Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to

  17. 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.

  18. Multiplexed Salivary Protein Profiling for Patients with Respiratory Diseases using Fiber-Optic Bundles and Fluorescent Antibody-Based Microarrays

    PubMed Central

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

    2013-01-01

    Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. Based on a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin 1 beta (IL-1β) salivary biomarkers in the sub-picomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p<0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis. PMID:23972398

  19. DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii

    DTIC Science & Technology

    2004-11-15

    DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii Chien-Chung Chao Rickettsiae Diseases...TITLE AND SUBTITLE DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii 5a. CONTRACT NUMBER 5b...ANSI Std Z39-18 Rickettsiae • Gram negative coccobacillary bacteria • Obligate intracellular organisms • Arthropod-borne • Cause febrile diseases (mild

  20. Transcriptomic profiling of long non-coding RNAs in dermatomyositis by microarray analysis

    PubMed Central

    Peng, Qing-Lin; Zhang, Ya-Mei; Yang, Han-Bo; Shu, Xiao-Ming; Lu, Xin; Wang, Guo-Chun

    2016-01-01

    Long non-coding RNAs (lncRNAs) are prevalently transcribed in the genome and have been found to be of functional importance. However, the potential roles of lncRNAs in dermatomyositis (DM) remain unknown. In this study, a lncRNA + mRNA microarray analysis was performed to profile lncRNAs and mRNAs from 15 treatment-naive DM patients and 5 healthy controls. We revealed a total of 1198 lncRNAs (322 up-regulated and 876 down-regulated) and 1213 mRNAs (665 up-regulated and 548 down-regulated) were significantly differentially expressed in DM patients compared with the healthy controls (fold change>2, P < 0.05). Subgrouping DM patients according to the presence of interstitial lung disease and anti-Jo-1 antibody revealed different expression patterns of the lncRNAs. Pathway and gene ontology analysis for the differentially expressed mRNAs confirmed that type 1 interferon signaling was the most significantly dysregulated pathway in all DM subgroups. In addition, distinct pathways that uniquely associated with DM subgroup were also identified. Bioinformatics prediction suggested that linc-DGCR6-1 may be a lncRNA that regulates type 1 interferon-inducible gene USP18, which was found highly expressed in the perifascicular areas of the muscle fibers of DM patients. Our findings provide an overview of aberrantly expressed lncRNAs in DM muscle and further broaden the understanding of DM pathogenesis. PMID:27605457

  1. Copasetic analysis: a framework for the blind analysis of microarray imagery.

    PubMed

    Fraser, K; O'Neill, P; Wang, Z; Liu, X

    2004-06-01

    From its conception, bioinformatics has been a multidisciplinary field which blends domain expert knowledge with new and existing processing techniques, all of which are focused on a common goal. Typically, these techniques have focused on the direct analysis of raw microarray image data. Unfortunately, this fails to utilise the image's full potential and in practice, this results in the lab technician having to guide the analysis algorithms. This paper presents a dynamic framework that aims to automate the process of microarray image analysis using a variety of techniques. An overview of the entire framework process is presented, the robustness of which is challenged throughout with a selection of real examples containing varying degrees of noise. The results show the potential of the proposed framework in its ability to determine slide layout accurately and perform analysis without prior structural knowledge. The algorithm achieves approximately, a 1 to 3 dB improved peak signal-to-noise ratio compared to conventional processing techniques like those implemented in GenePix when used by a trained operator. As far as the authors are aware, this is the first time such a comprehensive framework concept has been directly applied to the area of microarray image analysis.

  2. Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry

    PubMed Central

    Zhao, Jia; Patwa, Tasneem H.; Pal, Manoj; Qiu, Weilian; Lubman, David M.

    2010-01-01

    Summary Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin–streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis. PMID:19241043

  3. Microarray analysis of p-anisaldehyde-induced transcriptome of Saccharomyces cerevisiae.

    PubMed

    Yu, Lu; Guo, Na; Yang, Yi; Wu, Xiuping; Meng, Rizeng; Fan, Junwen; Ge, Fa; Wang, Xuelin; Liu, Jingbo; Deng, Xuming

    2010-03-01

    p-Anisaldehyde (4-methoxybenzaldehyde), an extract from Pimpinella anisum L. seeds, is a potential novel preservative. To reveal the possible action mechanism of p-anisaldehyde against microorganisms, yeast-based commercial oligonucleotide microarrays were used to analyze the genome-wide transcriptional changes in response to p-anisaldehyde. Quantitative real-time RT-PCR was performed for selected genes to verify the microarray results. We interpreted our microarray data with the clustering tool, T-profiler. Analysis of microarray data revealed that p-anisaldehyde induced the expression of genes related to sulphur assimilation, aromatic aldehydes metabolism, and secondary metabolism, which demonstrated that the addition of p-anisaldehyde may influence the normal metabolism of aromatic aldehydes. This genome-wide transcriptomics approach revealed first insights into the response of Saccharomyces cerevisiae (S. cerevisiae) to p-anisaldehyde challenge.

  4. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.

  5. THEME: a web tool for loop-design microarray data analysis.

    PubMed

    Chen, Chaang-Ray; Shu, Wun-Yi; Tsai, Min-Lung; Cheng, Wei-Chung; Hsu, Ian C

    2012-02-01

    A number of recent studies have shown that loop-design is more efficient than reference control design. Data analysis for loop-design microarray experiments is commonly undertaken using linear models and statistical tests. These techniques require specialized knowledge in statistical programming. However, limited loop-design web-based tools are available. We have developed the THEME (Tsing Hua Engine of Microarray Experiment) that exploits all necessary data analysis tools for loop-design microarray studies. THEME allows users to construct linear models and to apply multiple user-defined statistical tests of hypotheses for detection of DEG (differentially expressed genes). Users can modify entries of design matrix for experimental design as well as that of contrast matrix for statistical tests of hypotheses. The output of multiple user-defined statistical tests of hypotheses, DEG lists, can be cross-validated. The web platform provides data assessment and visualization tools that significantly assist users when evaluating the performance of microarray experimental procedures. THEME is also a MIAME (Minimal Information About a Microarray Experiment) compliant system, which enables users to export formatted files for GEO (Gene Expression Omnibus) submission. THEME offers comprehensive web services to biologists for data analysis of loop-design microarray experiments. This web-based resource is especially useful for core facility service as well as collaboration projects when researchers are not at the same site. Data analysis procedures, starting from uploading raw data files to retrieving DEG lists, can be flexibly operated with natural workflows. These features make THEME a reliable and powerful on-line system for data analysis of loop-design microarrays. The THEME server is available at http://metadb.bmes.nthu.edu.tw/theme/.

  6. Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

    PubMed Central

    Salisbury, Jesse; Hutchison, Keith W.; Wigglesworth, Karen; Eppig, John J.; Graber, Joel H.

    2009-01-01

    Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries. PMID:19834616

  7. Fully automated analysis of multi-resolution four-channel micro-array genotyping data

    NASA Astrophysics Data System (ADS)

    Abbaspour, Mohsen; Abugharbieh, Rafeef; Podder, Mohua; Tebbutt, Scott J.

    2006-03-01

    We present a fully-automated and robust microarray image analysis system for handling multi-resolution images (down to 3-micron with sizes up to 80 MBs per channel). The system is developed to provide rapid and accurate data extraction for our recently developed microarray analysis and quality control tool (SNP Chart). Currently available commercial microarray image analysis applications are inefficient, due to the considerable user interaction typically required. Four-channel DNA microarray technology is a robust and accurate tool for determining genotypes of multiple genetic markers in individuals. It plays an important role in the state of the art trend where traditional medical treatments are to be replaced by personalized genetic medicine, i.e. individualized therapy based on the patient's genetic heritage. However, fast, robust, and precise image processing tools are required for the prospective practical use of microarray-based genetic testing for predicting disease susceptibilities and drug effects in clinical practice, which require a turn-around timeline compatible with clinical decision-making. In this paper we have developed a fully-automated image analysis platform for the rapid investigation of hundreds of genetic variations across multiple genes. Validation tests indicate very high accuracy levels for genotyping results. Our method achieves a significant reduction in analysis time, from several hours to just a few minutes, and is completely automated requiring no manual interaction or guidance.

  8. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

    PubMed Central

    Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

    2015-01-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

  9. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

    PubMed

    Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

    2015-09-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

  10. Variance estimation in the analysis of microarray data.

    PubMed

    Wang, Yuedong; Ma, Yanyuan; Carroll, Raymond J

    2009-04-01

    Microarrays are one of the most widely used high throughput technologies. One of the main problems in the area is that conventional estimates of the variances that are required in the t-statistic and other statistics are unreliable owing to the small number of replications. Various methods have been proposed in the literature to overcome this lack of degrees of freedom problem. In this context, it is commonly observed that the variance increases proportionally with the intensity level, which has led many researchers to assume that the variance is a function of the mean. Here we concentrate on estimation of the variance as a function of an unknown mean in two models: the constant coefficient of variation model and the quadratic variance-mean model. Because the means are unknown and estimated with few degrees of freedom, naive methods that use the sample mean in place of the true mean are generally biased because of the errors-in-variables phenomenon. We propose three methods for overcoming this bias. The first two are variations on the theme of the so-called heteroscedastic simulation-extrapolation estimator, modified to estimate the variance function consistently. The third class of estimators is entirely different, being based on semiparametric information calculations. Simulations show the power of our methods and their lack of bias compared with the naive method that ignores the measurement error. The methodology is illustrated by using microarray data from leukaemia patients.

  11. Enhancing interdisciplinary mathematics and biology education: a microarray data analysis course bridging these disciplines.

    PubMed

    Tra, Yolande V; Evans, Irene M

    2010-01-01

    BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course.

  12. Enhancing Interdisciplinary Mathematics and Biology Education: A Microarray Data Analysis Course Bridging These Disciplines

    PubMed Central

    Evans, Irene M.

    2010-01-01

    BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course. PMID:20810954

  13. Genome-scale cluster analysis of replicated microarrays using shrinkage correlation coefficient

    PubMed Central

    Yao, Jianchao; Chang, Chunqi; Salmi, Mari L; Hung, Yeung Sam; Loraine, Ann; Roux, Stanley J

    2008-01-01

    Background Currently, clustering with some form of correlation coefficient as the gene similarity metric has become a popular method for profiling genomic data. The Pearson correlation coefficient and the standard deviation (SD)-weighted correlation coefficient are the two most widely-used correlations as the similarity metrics in clustering microarray data. However, these two correlations are not optimal for analyzing replicated microarray data generated by most laboratories. An effective correlation coefficient is needed to provide statistically sufficient analysis of replicated microarray data. Results In this study, we describe a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. The value of SCC is revealed by its comparison with two other correlation coefficients that are currently the most widely-used (Pearson correlation coefficient and SD-weighted correlation coefficient) using statistical measures on both synthetic expression data as well as real gene expression data from Saccharomyces cerevisiae. Two leading clustering methods, hierarchical and k-means clustering were applied for the comparison. The comparison indicated that using SCC achieves better clustering performance. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. Functional analysis suggested that some of the genetic mechanisms that control germination in such diverse plant lineages as mosses and angiosperms are also conserved among ferns. Conclusion This study shows that SCC is

  14. [Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays].

    PubMed

    Butvilovskaya, V I; Tsybulskaya, M V; Tikhonov, A A; Talibov, V O; Belousov, P V; Sazykin, A Yu; Schwartz, A M; Putlyaeva, L V; Surzhikov, S A; Stomakhin, A A; Solopova, O N; Rubina, A Yu

    2015-01-01

    The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.

  15. Microarray analysis for a comprehensive immunological-status evaluation during cancer vaccine immune monitoring.

    PubMed

    Monsurrò, Vladia; Marincola, Francesco M

    2011-01-01

    Anticancer immune responses can be enhanced by immune intervention that promotes complex biological mechanisms involving several cellular populations. The classical immune monitoring for biological-based cancer clinical trials is often based on single-cell analysis. However, the overall effect could be lost by such a reductionist approach explaining the lack of correlation among clinical and immunological endpoints often reported. Microarray technology could give the possibility of studying in a multiparametric setting the immune therapy effects. The application of microarray is leading to an improved understanding of the immune responses to tumor immunotherapy. In fact, analysis of cancer vaccine-induced host responses using microarrays is proposed as valuable alternative to the standard cell-based methods. This paper shows successful examples of how high-throughput gene expression profiling contributed to the understanding of anticancer immune responses during biological therapy, introducing as well the integrative platforms that allow the network analysis in molecular biology studies.

  16. Gene expression analysis of perennial ryegrass (Lolium perenne) using cDNA microarrays

    NASA Astrophysics Data System (ADS)

    Ong, Eng-Kok; Sawbridge, Tim; Webster, Tracie; Emmerling, Michael; Nguyen, Nga; Nunan, Katrina; O'Neill, Matthew; O'Toole, Fiona; Rhodes, Carolyn; Simmonds, Jason; Tian, Pei; Wearne, Katherine; Winkworth, Amanda; Spangenberg, German

    2003-07-01

    Perennial ryegrass (Lolium perenne) is a major forage grass of temperate pastures. A genomics program has been undertaken generating over 52,000 expressed sequence tags (ESTs). Cluster analysis of the ESTs identified approximately 14,600 ryegrass unigenes. In this report, we described the application of ryegrass unigene cDNAs to produce ryegrass 15K microarray. Fifteen microarray hybridisations were performed with labeled total RNA isolated from a variety of plant organs and developmental stages. In a proof of concept, gene expression profiling of ryegrass ESTs using the 15K unigene microarrays has been established using several known genes and two cluster analysis approaches (parallel coordinate planes plot and hierarchical clustering). The expression profile of the known genes (e.g. rubisco and invertase) corresponds well with published data. The microarray expression profile of a ryegrass putative root specific kinase gene was also verified with Northern blotting. This combination of DNA microarray hybridisations and cluster analysis can be applied as a tool for the identification of novel sequences of unknown function.

  17. Microarray analysis of potential genes in the pathogenesis of recurrent oral ulcer.

    PubMed

    Han, Jingying; He, Zhiwei; Li, Kun; Hou, Lu

    2015-01-01

    Recurrent oral ulcer seriously threatens patients' daily life and health. This study investigated potential genes and pathways that participate in the pathogenesis of recurrent oral ulcer by high throughput bioinformatic analysis. RT-PCR and Western blot were applied to further verify screened interleukins effect. Recurrent oral ulcer related genes were collected from websites and papers, and further found out from Human Genome 280 6.0 microarray data. Each pathway of recurrent oral ulcer related genes were got through chip hybridization. RT-PCR was applied to test four recurrent oral ulcer related genes to verify the microarray data. Data transformation, scatter plot, clustering analysis, and expression pattern analysis were used to analyze recurrent oral ulcer related gene expression changes. Recurrent oral ulcer gene microarray was successfully established. Microarray showed that 551 genes involved in recurrent oral ulcer activity and 196 genes were recurrent oral ulcer related genes. Of them, 76 genes up-regulated, 62 genes down-regulated, and 58 genes up-/down-regulated. Total expression level up-regulated 752 times (60%) and down-regulated 485 times (40%). IL-2 plays an important role in the occurrence, development and recurrence of recurrent oral ulcer on the mRNA and protein levels. Gene microarray can be used to analyze potential genes and pathways in recurrent oral ulcer. IL-2 may be involved in the pathogenesis of recurrent oral ulcer.

  18. Microarray analysis of thioacetamide-treated type 1 diabetic rats

    SciTech Connect

    Devi, Sachin S.; Mehendale, Harihara M. . E-mail: mehendale@ulm.edu

    2006-04-01

    It is well known that diabetes imparts high sensitivity to numerous hepatotoxicants. Previously, we have shown that a normally non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats due to inhibited tissue repair allowing progression of liver injury. On the other hand, DB rats exposed to 30 mg TA/kg exhibit delayed tissue repair and delayed recovery from injury. The objective of this study was to investigate the mechanism of impaired tissue repair and progression of liver injury in TA-treated DB rats by using cDNA microarray. Gene expression pattern was examined at 0, 6, and 12 h after TA challenge, and selected mechanistic leads from microarray experiments were confirmed by real-time RT-PCR and further investigated at protein level over the time course of 0 to 36 h after TA treatment. Diabetic condition itself increased gene expression of proteases and decreased gene expression of protease inhibitors. Administration of 300 mg TA/kg to DB rats further elevated gene expression of proteases and suppressed gene expression of protease inhibitors, explaining progression of liver injury in DB rats after TA treatment. Inhibited expression of genes involved in cell division cycle (cyclin D1, IGFBP-1, ras, E2F) was observed after exposure of DB rats to 300 mg TA/kg, explaining inhibited tissue repair in these rats. On the other hand, DB rats receiving 30 mg TA/kg exhibit delayed expression of genes involved in cell division cycle, explaining delayed tissue repair in these rats. In conclusion, impaired cyclin D1 signaling along with increased proteases and decreased protease inhibitors may explain impaired tissue repair that leads to progression of liver injury initiated by TA in DB rats.

  19. Microarray analysis of circular RNA expression patterns in polarized macrophages

    PubMed Central

    Zhang, Yingying; Zhang, Yao; Li, Xueqin; Zhang, Mengying; Lv, Kun

    2017-01-01

    Circular RNAs (circRNAs) are generated from diverse genomic locations and are a new player in the regulation of post-transcriptional gene expression. Recent studies have revealed that circRNAs play a crucial role in fine-tuning the level of microRNA (miRNA)-mediated regulation of gene expression by sequestering miRNAs. The interaction of circRNAs with disease-associated miRNAs suggests that circRNAs are important in the pathology of disease. However, the effects and roles of circRNAs in macrophage polarization have yet to be explored. In the present study, we performed a circRNA microarray to compare the circRNA expression profiles of bone marrow-derived macrophages (BMDMs) under two distinct polarizing conditions (M1 macrophages induced by interferon-γ and LPS stimulation, and M2 macrophages induced by interleukin-4 stimulation). Our results showed that a total of 189 circRNAs were differentially expressed between M1 and M2 macrophages. Differentially expressed circRNAs with a high fold-change were selected for validation by RT-qPCR: circRNA-003780, circRNA-010056, and circRNA-010231 were upregulated and circRNA-003424, circRNA-013630, circRNA-001489 and circRNA-018127 were downregulated (fold-change >4, P<0.05) in M1 compared to M2, which was found to correlate with the microarray data. Furthermore, the most differentially expressed circRNAs within all the comparisons were annotated in detail with circRNA/miRNA interaction information using miRNA target prediction software. In conclusion, the present study provides novel insight into the role of circRNAs in macrophage differentiation and polarization. PMID:28075448

  20. Characterizing Antibody Responses to Plasmodium vivax and Plasmodium falciparum Antigens in India Using Genome-Scale Protein Microarrays.

    PubMed

    Uplekar, Swapna; Rao, Pavitra Nagesh; Ramanathapuram, Lalitha; Awasthi, Vikky; Verma, Kalpana; Sutton, Patrick; Ali, Syed Zeeshan; Patel, Ankita; G, Sri Lakshmi Priya; Ravishankaran, Sangamithra; Desai, Nisha; Tandel, Nikunj; Choubey, Sandhya; Barla, Punam; Kanagaraj, Deena; Eapen, Alex; Pradhan, Khageswar; Singh, Ranvir; Jain, Aarti; Felgner, Philip L; Davies, D Huw; Carlton, Jane M; Das, Jyoti

    2017-01-01

    Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.

  1. Characterizing Antibody Responses to Plasmodium vivax and Plasmodium falciparum Antigens in India Using Genome-Scale Protein Microarrays

    PubMed Central

    Awasthi, Vikky; Verma, Kalpana; Sutton, Patrick; Ali, Syed Zeeshan; Patel, Ankita; G., Sri Lakshmi Priya; Ravishankaran, Sangamithra; Desai, Nisha; Tandel, Nikunj; Choubey, Sandhya; Barla, Punam; Kanagaraj, Deena; Eapen, Alex; Pradhan, Khageswar; Singh, Ranvir; Jain, Aarti; Felgner, Philip L.; Davies, D. Huw; Das, Jyoti

    2017-01-01

    Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world. PMID:28118367

  2. 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.

  3. A probabilistic framework for microarray data analysis: fundamental probability models and statistical inference.

    PubMed

    Ogunnaike, Babatunde A; Gelmi, Claudio A; Edwards, Jeremy S

    2010-05-21

    Gene expression studies generate large quantities of data with the defining characteristic that the number of genes (whose expression profiles are to be determined) exceed the number of available replicates by several orders of magnitude. Standard spot-by-spot analysis still seeks to extract useful information for each gene on the basis of the number of available replicates, and thus plays to the weakness of microarrays. On the other hand, because of the data volume, treating the entire data set as an ensemble, and developing theoretical distributions for these ensembles provides a framework that plays instead to the strength of microarrays. We present theoretical results that under reasonable assumptions, the distribution of microarray intensities follows the Gamma model, with the biological interpretations of the model parameters emerging naturally. We subsequently establish that for each microarray data set, the fractional intensities can be represented as a mixture of Beta densities, and develop a procedure for using these results to draw statistical inference regarding differential gene expression. We illustrate the results with experimental data from gene expression studies on Deinococcus radiodurans following DNA damage using cDNA microarrays.

  4. Gene ARMADA: an integrated multi-analysis platform for microarray data implemented in MATLAB

    PubMed Central

    Chatziioannou, Aristotelis; Moulos, Panagiotis; Kolisis, Fragiskos N

    2009-01-01

    Background The microarray data analysis realm is ever growing through the development of various tools, open source and commercial. However there is absence of predefined rational algorithmic analysis workflows or batch standardized processing to incorporate all steps, from raw data import up to the derivation of significantly differentially expressed gene lists. This absence obfuscates the analytical procedure and obstructs the massive comparative processing of genomic microarray datasets. Moreover, the solutions provided, heavily depend on the programming skills of the user, whereas in the case of GUI embedded solutions, they do not provide direct support of various raw image analysis formats or a versatile and simultaneously flexible combination of signal processing methods. Results We describe here Gene ARMADA (Automated Robust MicroArray Data Analysis), a MATLAB implemented platform with a Graphical User Interface. This suite integrates all steps of microarray data analysis including automated data import, noise correction and filtering, normalization, statistical selection of differentially expressed genes, clustering, classification and annotation. In its current version, Gene ARMADA fully supports 2 coloured cDNA and Affymetrix oligonucleotide arrays, plus custom arrays for which experimental details are given in tabular form (Excel spreadsheet, comma separated values, tab-delimited text formats). It also supports the analysis of already processed results through its versatile import editor. Besides being fully automated, Gene ARMADA incorporates numerous functionalities of the Statistics and Bioinformatics Toolboxes of MATLAB. In addition, it provides numerous visualization and exploration tools plus customizable export data formats for seamless integration by other analysis tools or MATLAB, for further processing. Gene ARMADA requires MATLAB 7.4 (R2007a) or higher and is also distributed as a stand-alone application with MATLAB Component Runtime

  5. Improved covariance matrix estimators for weighted analysis of microarray data.

    PubMed

    Astrand, Magnus; Mostad, Petter; Rudemo, Mats

    2007-12-01

    Empirical Bayes models have been shown to be powerful tools for identifying differentially expressed genes from gene expression microarray data. An example is the WAME model, where a global covariance matrix accounts for array-to-array correlations as well as differing variances between arrays. However, the existing method for estimating the covariance matrix is very computationally intensive and the estimator is biased when data contains many regulated genes. In this paper, two new methods for estimating the covariance matrix are proposed. The first method is a direct application of the EM algorithm for fitting the multivariate t-distribution of the WAME model. In the second method, a prior distribution for the log fold-change is added to the WAME model, and a discrete approximation is used for this prior. Both methods are evaluated using simulated and real data. The first method shows equal performance compared to the existing method in terms of bias and variability, but is superior in terms of computer time. For large data sets (>15 arrays), the second method also shows superior computer run time. Moreover, for simulated data with regulated genes the second method greatly reduces the bias. With the proposed methods it is possible to apply the WAME model to large data sets with reasonable computer run times. The second method shows a small bias for simulated data, but appears to have a larger bias for real data with many regulated genes.

  6. Microarray Analysis of the Microflora of Root Caries in Elderly

    PubMed Central

    Preza, Dorita; Olsen, Ingar; Willumsen, Tiril; Boches, Susan K.; Cotton, Sean L.; Grinde, Bjørn; Paster, Bruce J.

    2009-01-01

    Purpose The present study used a new 16S rRNA-based microarray with probes for over 300 bacterial species better define the bacterial profiles of healthy root surfaces and root caries (RC) in the elderly. Materials Supragingival plaque was collected from 20 healthy subjects (Controls) and from healthy and carious roots and carious dentin from 21 RC subjects (Patients). Results Collectively, 179 bacterial species and species groups were detected. A higher bacterial diversity was observed in the Controls as compared to Patients. Lactobacillus casei/paracasei/rhamnosus and Pseudoramibacter alactolyticus were notably associated with most root caries samples. Streptococcus mutans was detected more frequently in the infected dentin than in the other samples, but the difference was not significant. Actinomyces were found more frequently in Controls. Conclusion Actinomyces and S. mutans may play a limited role as pathogens of RC. The results from this study were in agreement with those of our previous study based on 16S rRNA gene sequencing with 72% of the species being detected with both methods. PMID:19039610

  7. A protein microarray-based analysis of S-nitrosylation

    PubMed Central

    Foster, Matthew W.; Forrester, Michael T.; Stamler, Jonathan S.

    2009-01-01

    The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of α-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols. PMID:19864628

  8. A protein microarray-based analysis of S-nitrosylation.

    PubMed

    Foster, Matthew W; Forrester, Michael T; Stamler, Jonathan S

    2009-11-10

    The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.

  9. Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

    PubMed Central

    Huyghe, Antoine; François, Patrice; Mombelli, Andrea; Tangomo, Manuela; Girard, Myriam; Baratti-Mayer, Denise; Bolivar, Ignacio; Pittet, Didier; Schrenzel, Jacques

    2013-01-01

    Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma. PMID:24086784

  10. Microarray and KOG analysis of Acanthamoeba healyi genes up-regulated by mouse-brain passage.

    PubMed

    Moon, Eun-Kyung; Xuan, Ying-Hua; Kong, Hyun-Hee

    2014-08-01

    Long-term cultivation in a laboratory could reduce the virulence of Acanthamoeba. To identify virulence factors of Acanthamoeba, the authors compared the transcription profiles of long-term cultivated Acanthamoeba healyi (OLD) and three times mouse-brain passaged A. healyi (MBP) using microarray analysis and eukaryotic orthologous group (KOG) assignments. Microarray analysis revealed that 601 genes were up-regulated by mouse-brain passage. The results of real-time PCR of 8 randomly selected genes up-regulated in the MBP strain confirmed microarray analysis findings. KOG assignments showed relatively higher percentages of the MBP strain up-regulated genes in T article (signal transduction mechanism), O article (posttranslational modification, protein turnover, chaperones), C article (energy production and conversion), and J article (translation, ribosomal structure and biogenesis). In particular, the MBP strain showed higher expressions of cysteine protease and metalloprotease. A comparison of KOG assignments by microarray analysis and previous EST (expressed sequence tags) analysis showed similar populations of up-regulated genes. These results provide important information regarding the identification of virulence factors of pathogenic Acanthamoeba.

  11. Microarray Meta-Analysis and Cross-Platform Normalization: Integrative Genomics for Robust Biomarker Discovery

    PubMed Central

    Walsh, Christopher J.; Hu, Pingzhao; Batt, Jane; Dos Santos, Claudia C.

    2015-01-01

    The diagnostic and prognostic potential of the vast quantity of publicly-available microarray data has driven the development of methods for integrating the data from different microarray platforms. Cross-platform integration, when appropriately implemented, has been shown to improve reproducibility and robustness of gene signature biomarkers. Microarray platform integration can be conceptually divided into approaches that perform early stage integration (cross-platform normalization) versus late stage data integration (meta-analysis). A growing number of statistical methods and associated software for platform integration are available to the user, however an understanding of their comparative performance and potential pitfalls is critical for best implementation. In this review we provide evidence-based, practical guidance to researchers performing cross-platform integration, particularly with an objective to discover biomarkers. PMID:27600230

  12. Identification of key genes associated with cervical cancer by comprehensive analysis of transcriptome microarray and methylation microarray

    PubMed Central

    LIU, MING-YAN; ZHANG, HONG; HU, YUAN-JING; CHEN, YU-WEI; ZHAO, XIAO-NAN

    2016-01-01

    Cervical cancer is the second most commonly diagnosed type of cancer and the third leading cause of cancer-associated mortality in women. The current study aimed to determine the genes associated with cervical cancer development. Microarray data (GSE55940 and GSE46306) were downloaded from Gene Expression Omnibus. Overlapping genes between the differentially expressed genes (DEGs) in GSE55940 (identified by Limma package) and the differentially methylated genes were screened. Gene Ontology (GO) enrichment analysis was subsequently performed for these genes using the ToppGene database. In GSE55940, 91 downregulated and 151 upregulated DEGs were identified. In GSE46306, 561 overlapping differentially methylated genes were obtained through the differential methylation analysis at the CpG site level, CpG island level and gene level. A total of 5 overlapping genes [dipeptidyl peptidase 4 (DPP4); endothelin 3 (EDN3); fibroblast growth factor 14 (FGF14); tachykinin, precursor 1 (TAC1); and wingless-type MMTV integration site family, member 16 (WNT16)] between the 561 overlapping differentially methylated genes and the 242 DEGs were identified, which were downregulated and hypermethylated simultaneously in cervical cancer samples. Enriched GO terms were receptor binding (involving DPP4, EDN3, FGF14, TAC1 and WNT16), ameboidal-type cell migration (DPP4, EDN3 and TAC1), mitogen-activated protein kinase cascade (FGF14, EDN3 and WNT16) and cell proliferation (EDN3, WNT16, DPP4 and TAC1). These results indicate that DPP4, EDN3, FGF14, TAC1 and WNT16 may be involved in the pathogenesis of cervical cancer. PMID:27347167

  13. Enhancing Interdisciplinary Mathematics and Biology Education: A Microarray Data Analysis Course Bridging These Disciplines

    ERIC Educational Resources Information Center

    Tra, Yolande V.; Evans, Irene M.

    2010-01-01

    "BIO2010" put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on…

  14. The Utility of Chromosomal Microarray Analysis in Developmental and Behavioral Pediatrics

    ERIC Educational Resources Information Center

    Beaudet, Arthur L.

    2013-01-01

    Chromosomal microarray analysis (CMA) has emerged as a powerful new tool to identify genomic abnormalities associated with a wide range of developmental disabilities including congenital malformations, cognitive impairment, and behavioral abnormalities. CMA includes array comparative genomic hybridization (CGH) and single nucleotide polymorphism…

  15. MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    EPA Science Inventory


    MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

  16. Diagnostic Yield of Chromosomal Microarray Analysis in an Autism Primary Care Practice: Which Guidelines to Implement?

    ERIC Educational Resources Information Center

    McGrew, Susan G.; Peters, Brittany R.; Crittendon, Julie A.; Veenstra-VanderWeele, Jeremy

    2012-01-01

    Genetic testing is recommended for patients with ASD; however specific recommendations vary by specialty. American Academy of Pediatrics and American Academy of Neurology guidelines recommend G-banded karyotype and Fragile X DNA. The American College of Medical Genetics recommends Chromosomal Microarray Analysis (CMA). We determined the yield of…

  17. Parents' Perceptions of the Usefulness of Chromosomal Microarray Analysis for Children with Autism Spectrum Disorders

    ERIC Educational Resources Information Center

    Reiff, Marian; Giarelli, Ellen; Bernhardt, Barbara A.; Easley, Ebony; Spinner, Nancy B.; Sankar, Pamela L.; Mulchandani, Surabhi

    2015-01-01

    Clinical guidelines recommend chromosomal microarray analysis (CMA) for all children with autism spectrum disorders (ASDs). We explored the test's perceived usefulness among parents of children with ASD who had undergone CMA, and received a result categorized as pathogenic, variant of uncertain significance, or negative. Fifty-seven parents…

  18. 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

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

    SciTech Connect

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

    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) 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.

  20. Array2BIO: A Comprehensive Suite of Utilities for the Analysis of Microarray Data

    SciTech Connect

    Loots, G G; Chain, P G; Mabery, S; Rasley, A; Garcia, E; Ovcharenko, I

    2006-02-13

    We have developed an integrative and automated toolkit for the analysis of Affymetrix microarray data, named Array2BIO. It identifies groups of coexpressed genes using two complementary approaches--comparative analysis of signal versus control microarrays and clustering analysis of gene expression across different conditions. The identified genes are assigned to functional categories based on the Gene Ontology classification, and a detection of corresponding KEGG protein interaction pathways. Array2BIO reliably handles low-expressor genes and provides a set of statistical methods to quantify the odds of observations, including the Benjamini-Hochberg and Bonferroni multiple testing corrections. Automated interface with the ECR Browser provides evolutionary conservation analysis of identified gene loci while the interconnection with Creme allows high-throughput analysis of human promoter regions and prediction of gene regulatory elements that underlie the observed expression patterns. Array2BIO is publicly available at http://array2bio.dcode.org.

  1. 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.

  2. Informatics Enhanced SNP Microarray Analysis of 30 Miscarriage Samples Compared to Routine Cytogenetics

    PubMed Central

    Lathi, Ruth B.; Loring, Megan; Massie, Jamie A. M.; Demko, Zachary P.; Johnson, David; Sigurjonsson, Styrmir; Gemelos, George; Rabinowitz, Matthew

    2012-01-01

    Purpose The metaphase karyotype is often used as a diagnostic tool in the setting of early miscarriage; however this technique has several limitations. We evaluate a new technique for karyotyping that uses single nucleotide polymorphism microarrays (SNP). This technique was compared in a blinded, prospective fashion, to the traditional metaphase karyotype. Methods Patients undergoing dilation and curettage for first trimester miscarriage between February and August 2010 were enrolled. Samples of chorionic villi were equally divided and sent for microarray testing in parallel with routine cytogenetic testing. Results Thirty samples were analyzed, with only four discordant results. Discordant results occurred when the entire genome was duplicated or when a balanced rearrangement was present. Cytogenetic karyotyping took an average of 29 days while microarray-based karytoyping took an average of 12 days. Conclusions Molecular karyotyping of POC after missed abortion using SNP microarray analysis allows for the ability to detect maternal cell contamination and provides rapid results with good concordance to standard cytogenetic analysis. PMID:22403611

  3. Gene microarray data analysis using parallel point-symmetry-based clustering.

    PubMed

    Sarkar, Anasua; Maulik, Ujjwal

    2015-01-01

    Identification of co-expressed genes is the central goal in microarray gene expression analysis. Point-symmetry-based clustering is an important unsupervised learning technique for recognising symmetrical convex- or non-convex-shaped clusters. To enable fast clustering of large microarray data, we propose a distributed time-efficient scalable approach for point-symmetry-based K-Means algorithm. A natural basis for analysing gene expression data using symmetry-based algorithm is to group together genes with similar symmetrical expression patterns. This new parallel implementation also satisfies linear speedup in timing without sacrificing the quality of clustering solution on large microarray data sets. The parallel point-symmetry-based K-Means algorithm is compared with another new parallel symmetry-based K-Means and existing parallel K-Means over eight artificial and benchmark microarray data sets, to demonstrate its superiority, in both timing and validity. The statistical analysis is also performed to establish the significance of this message-passing-interface based point-symmetry K-Means implementation. We also analysed the biological relevance of clustering solutions.

  4. Analysis of gene expression on anodic porous alumina microarrays

    PubMed Central

    Nicolini, Claudio; Singh, Manjul; Spera, Rosanna; Felli, Lamberto

    2013-01-01

    This paper investigates the application of anodic porous alumina as an advancement on chip laboratory for gene expressions. The surface was prepared by a suitable electrolytic process to obtain a regular distribution of deep micrometric holes and printed bypen robot tips under standard conditions. The gene expression within the Nucleic Acid Programmable Protein Array (NAPPA) is realized in a confined environment of 16 spots, containing circular DNA plasmids expressed using rabbit reticulocyte lysate. Authors demonstrated the usefulness of APA in withholding the protein expression by detecting with a CCD microscope the photoluminescence signal emitted from the complex secondary antibody anchored to Cy3 and confined in the pores. Friction experiments proved the mechanical resistance under external stresses by the robot tip pens printing. So far, no attempts have been made to directly compare APA with any other surface/substrate; the rationale for pursuing APA as a potential surface coating is that it provides advantages over the simple functionalization of a glass slide, overcoming concerns about printing and its ability to generate viable arrays. PMID:23783000

  5. Comparative analysis of gene expression by microarray analysis of male and female flowers of Asparagus officinalis.

    PubMed

    Gao, Wu-Jun; Li, Shu-Fen; Zhang, Guo-Jun; Wang, Ning-Na; Deng, Chuan-Liang; Lu, Long-Dou

    2013-01-01

    To identify rapidly a number of genes probably involved in sex determination and differentiation of the dioecious plant Asparagus officinalis, gene expression profiles in early flower development for male and female plants were investigated by microarray assay with 8,665 probes. In total, 638 male-biased and 543 female-biased genes were identified. These genes with biased-expression for male and female were involved in a variety of processes associated with molecular functions, cellular components, and biological processes, suggesting that a complex mechanism underlies the sex development of asparagus. Among the differentially expressed genes involved in the reproductive process, a number of genes associated with floral development were identified. Reverse transcription-PCR was performed for validation, and the results were largely consistent with those obtained by microarray analysis. The findings of this study might contribute to understanding of the molecular mechanisms of sex determination and differentiation in dioecious asparagus and provide a foundation for further studies of this plant.

  6. Genomic Imbalances in Neonates With Birth Defects: High Detection Rates by Using Chromosomal Microarray Analysis

    PubMed Central

    Lu, Xin-Yan; Phung, Mai T.; Shaw, Chad A.; Pham, Kim; Neil, Sarah E.; Patel, Ankita; Sahoo, Trilochan; Bacino, Carlos A.; Stankiewicz, Pawel; Lee Kang, Sung-Hae; Lalani, Seema; Chinault, A. Craig; Lupski, James R.; Cheung, Sau W.; Beaudet, Arthur L.

    2009-01-01

    OBJECTIVES Our aim was to determine the frequency of genomic imbalances in neonates with birth defects by using targeted array-based comparative genomic hybridization, also known as chromosomal microarray analysis. METHODS Between March 2006 and September 2007, 638 neonates with various birth defects were referred for chromosomal microarray analysis. Three consecutive chromosomal microarray analysis versions were used: bacterial artificial chromosome-based versions V5 and V6 and bacterial artificial chromosome emulated oligonucleotide-based version V6 Oligo. Each version had targeted but increasingly extensive genomic coverage and interrogated >150 disease loci with enhanced coverage in genomic rearrangement-prone pericentromeric and subtelomeric regions. RESULTS Overall, 109 (17.1%) patients were identified with clinically significant abnormalities with detection rates of 13.7%, 16.6%, and 19.9% on V5, V6, and V6 Oligo, respectively. The majority of these abnormalities would not be defined by using karyotype analysis. The clinically significant detection rates by use of chromosomal microarray analysis for various clinical indications were 66.7% for “possible chromosomal abnormality” ± “others” (other clinical indications), 33.3% for ambiguous genitalia ± others, 27.1% for dysmorphic features + multiple congenital anomalies ± others, 24.6% for dysmorphic features ± others, 21.8% for congenital heart disease ± others, 17.9% for multiple congenital anomalies ± others, and 9.5% for the patients referred for others that were different from the groups defined. In all, 16 (2.5%) patients had chromosomal aneuploidies, and 81 (12.7%) patients had segmental aneusomies including common microdeletion or microduplication syndromes and other genomic disorders. Chromosomal mosaicism was found in 12 (1.9%) neonates. CONCLUSIONS Chromosomal microarray analysis is a valuable clinical diagnostic tool that allows precise and rapid identification of genomic imbalances

  7. Knowledge-based analysis of microarrays for the discovery of transcriptional regulation relationships

    PubMed Central

    2010-01-01

    Background The large amount of high-throughput genomic data has facilitated the discovery of the regulatory relationships between transcription factors and their target genes. While early methods for discovery of transcriptional regulation relationships from microarray data often focused on the high-throughput experimental data alone, more recent approaches have explored the integration of external knowledge bases of gene interactions. Results In this work, we develop an algorithm that provides improved performance in the prediction of transcriptional regulatory relationships by supplementing the analysis of microarray data with a new method of integrating information from an existing knowledge base. Using a well-known dataset of yeast microarrays and the Yeast Proteome Database, a comprehensive collection of known information of yeast genes, we show that knowledge-based predictions demonstrate better sensitivity and specificity in inferring new transcriptional interactions than predictions from microarray data alone. We also show that comprehensive, direct and high-quality knowledge bases provide better prediction performance. Comparison of our results with ChIP-chip data and growth fitness data suggests that our predicted genome-wide regulatory pairs in yeast are reasonable candidates for follow-up biological verification. Conclusion High quality, comprehensive, and direct knowledge bases, when combined with appropriate bioinformatic algorithms, can significantly improve the discovery of gene regulatory relationships from high throughput gene expression data. PMID:20122245

  8. Microarray analysis of gene expression during early development: a cautionary overview.

    PubMed

    Robert, Claude

    2010-12-01

    The rise of the 'omics' technologies started nearly a decade ago and, among them, transcriptomics has been used successfully to contrast gene expression in mammalian oocytes and early embryos. The scarcity of biological material that early developmental stages provide is the prime reason why the field of transcriptomics is becoming more and more popular with reproductive biologists. The potential to amplify scarce mRNA samples and generate the necessary amounts of starting material enables the relative measurement of RNA abundance of thousands of candidates simultaneously. So far, microarrays have been the most commonly used high-throughput method in this field. Microarray platforms can be found in a wide variety of formats, from cDNA collections to long or short oligo probe sets. These platforms generate large amounts of data that require the integration of comparative RNA abundance values in the physiological context of early development for their full benefit to be appreciated. Unfortunately, significant discrepancies between datasets suggest that direct comparison between studies is difficult and often not possible. We have investigated the sample-handling steps leading to the generation of microarray data produced from prehatching embryo samples and have identified key steps that significantly impact the downstream results. This review provides a discussion on the best methods for the preparation of samples from early embryos for microarray analysis and focuses on the challenges that impede dataset comparisons from different platforms and the reasons why methodological benchmarking performed using somatic cells may not apply to the atypical nature of prehatching development.

  9. Comprehensive analysis of correlation coefficients estimated from pooling heterogeneous microarray data

    PubMed Central

    2013-01-01

    Background The synthesis of information across microarray studies has been performed by combining statistical results of individual studies (as in a mosaic), or by combining data from multiple studies into a large pool to be analyzed as a single data set (as in a melting pot of data). Specific issues relating to data heterogeneity across microarray studies, such as differences within and between labs or differences among experimental conditions, could lead to equivocal results in a melting pot approach. Results We applied statistical theory to determine the specific effect of different means and heteroskedasticity across 19 groups of microarray data on the sign and magnitude of gene-to-gene Pearson correlation coefficients obtained from the pool of 19 groups. We quantified the biases of the pooled coefficients and compared them to the biases of correlations estimated by an effect-size model. Mean differences across the 19 groups were the main factor determining the magnitude and sign of the pooled coefficients, which showed largest values of bias as they approached ±1. Only heteroskedasticity across the pool of 19 groups resulted in less efficient estimations of correlations than did a classical meta-analysis approach of combining correlation coefficients. These results were corroborated by simulation studies involving either mean differences or heteroskedasticity across a pool of N > 2 groups. Conclusions The combination of statistical results is best suited for synthesizing the correlation between expression profiles of a gene pair across several microarray studies. PMID:23822712

  10. GEPAS, a web-based tool for microarray data analysis and interpretation

    PubMed Central

    Tárraga, Joaquín; Medina, Ignacio; Carbonell, José; Huerta-Cepas, Jaime; Minguez, Pablo; Alloza, Eva; Al-Shahrour, Fátima; Vegas-Azcárate, Susana; Goetz, Stefan; Escobar, Pablo; Garcia-Garcia, Francisco; Conesa, Ana; Montaner, David; Dopazo, Joaquín

    2008-01-01

    Gene Expression Profile Analysis Suite (GEPAS) is one of the most complete and extensively used web-based packages for microarray data analysis. During its more than 5 years of activity it has continuously been updated to keep pace with the state-of-the-art in the changing microarray data analysis arena. GEPAS offers diverse analysis options that include well established as well as novel algorithms for normalization, gene selection, class prediction, clustering and functional profiling of the experiment. New options for time-course (or dose-response) experiments, microarray-based class prediction, new clustering methods and new tests for differential expression have been included. The new pipeliner module allows automating the execution of sequential analysis steps by means of a simple but powerful graphic interface. An extensive re-engineering of GEPAS has been carried out which includes the use of web services and Web 2.0 technology features, a new user interface with persistent sessions and a new extended database of gene identifiers. GEPAS is nowadays the most quoted web tool in its field and it is extensively used by researchers of many countries and its records indicate an average usage rate of 500 experiments per day. GEPAS, is available at http://www.gepas.org. PMID:18508806

  11. Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

    PubMed Central

    2010-01-01

    Background Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. Results We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. Conclusions MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions. PMID:20509979

  12. A rapid automatic processing platform for bead label-assisted microarray analysis: application for genetic hearing-loss mutation detection.

    PubMed

    Zhu, Jiang; Song, Xiumei; Xiang, Guangxin; Feng, Zhengde; Guo, Hongju; Mei, Danyang; Zhang, Guohao; Wang, Dong; Mitchelson, Keith; Xing, Wanli; Cheng, Jing

    2014-04-01

    Molecular diagnostics using microarrays are increasingly being used in clinical diagnosis because of their high throughput, sensitivity, and accuracy. However, standard microarray processing takes several hours and involves manual steps during hybridization, slide clean up, and imaging. Here we describe the development of an integrated platform that automates these individual steps as well as significantly shortens the processing time and improves reproducibility. The platform integrates such key elements as a microfluidic chip, flow control system, temperature control system, imaging system, and automated analysis of clinical results. Bead labeling of microarray signals required a simple imaging system and allowed continuous monitoring of the microarray processing. To demonstrate utility, the automated platform was used to genotype hereditary hearing-loss gene mutations. Compared with conventional microarray processing procedures, the platform increases the efficiency and reproducibility of hybridization, speeding microarray processing through to result analysis. The platform also continuously monitors the microarray signals, which can be used to facilitate optimization of microarray processing conditions. In addition, the modular design of the platform lends itself to development of simultaneous processing of multiple microfluidic chips. We believe the novel features of the platform will benefit its use in clinical settings in which fast, low-complexity molecular genetic testing is required.

  13. Microarray analysis of differentially expressed genes between cysts and trophozoites of Acanthamoeba castellanii.

    PubMed

    Moon, Eun-Kyung; Xuan, Ying-Hua; Chung, Dong-Il; Hong, Yeonchul; Kong, Hyun-Hee

    2011-12-01

    Acanthamoeba infection is difficult to treat because of the resistance property of Acanthamoeba cyst against the host immune system, diverse antibiotics, and therapeutic agents. To identify encystation mediating factors of Acanthamoeba, we compared the transcription profile between cysts and trophozoites using microarray analysis. The DNA chip was composed of 12,544 genes based on expressed sequence tag (EST) from an Acanthamoeba ESTs database (DB) constructed in our laboratory, genetic information of Acanthamoeba from TBest DB, and all of Acanthamoeba related genes registered in the NCBI. Microarray analysis indicated that 701 genes showed higher expression than 2 folds in cysts than in trophozoites, and 859 genes were less expressed in cysts than in trophozoites. The results of real-time PCR analysis of randomly selected 9 genes of which expression was increased during cyst formation were coincided well with the microarray results. Eukaryotic orthologous groups (KOG) analysis showed an increment in T article (signal transduction mechanisms) and O article (posttranslational modification, protein turnover, and chaperones) whereas significant decrement of C article (energy production and conversion) during cyst formation. Especially, cystein proteinases showed high expression changes (282 folds) with significant increases in real-time PCR, suggesting a pivotal role of this proteinase in the cyst formation of Acanthamoeba. The present study provides important clues for the identification and characterization of encystation mediating factors of Acanthamoeba.

  14. Systematic analysis of microarray datasets to identify Parkinson's disease-associated pathways and genes

    PubMed Central

    Feng, Yinling; Wang, Xuefeng

    2017-01-01

    In order to investigate commonly disturbed genes and pathways in various brain regions of patients with Parkinson's disease (PD), microarray datasets from previous studies were collected and systematically analyzed. Different normalization methods were applied to microarray datasets from different platforms. A strategy combining gene co-expression networks and clinical information was adopted, using weighted gene co-expression network analysis (WGCNA) to screen for commonly disturbed genes in different brain regions of patients with PD. Functional enrichment analysis of commonly disturbed genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Co-pathway relationships were identified with Pearson's correlation coefficient tests and a hypergeometric distribution-based test. Common genes in pathway pairs were selected out and regarded as risk genes. A total of 17 microarray datasets from 7 platforms were retained for further analysis. Five gene coexpression modules were identified, containing 9,745, 736, 233, 101 and 93 genes, respectively. One module was significantly correlated with PD samples and thus the 736 genes it contained were considered to be candidate PD-associated genes. Functional enrichment analysis demonstrated that these genes were implicated in oxidative phosphorylation and PD. A total of 44 pathway pairs and 52 risk genes were revealed, and a risk gene pathway relationship network was constructed. Eight modules were identified and were revealed to be associated with PD, cancers and metabolism. A number of disturbed pathways and risk genes were unveiled in PD, and these findings may help advance understanding of PD pathogenesis. PMID:28098893

  15. Application of phylogenetic microarray analysis to discriminate sources of fecal pollution.

    PubMed

    Dubinsky, Eric A; Esmaili, Laleh; Hulls, John R; Cao, Yiping; Griffith, John F; Andersen, Gary L

    2012-04-17

    Conventional methods for fecal source tracking typically use single biomarkers to systematically identify or exclude sources. High-throughput DNA sequence analysis can potentially identify all sources of microbial contaminants in a single test by measuring the total diversity of fecal microbial communities. In this study, we used phylogenetic microarray analysis to determine the comprehensive suite of bacteria that define major sources of fecal contamination in coastal California. Fecal wastes were collected from 42 different populations of humans, birds, cows, horses, elk, and pinnipeds. We characterized bacterial community composition using a DNA microarray that probes for 16S rRNA genes of 59,316 different bacterial taxa. Cluster analysis revealed strong differences in community composition among fecal wastes from human, birds, pinnipeds, and grazers. Actinobacteria, Bacilli, and many Gammaproteobacteria taxa discriminated birds from mammalian sources. Diverse families within the Clostridia and Bacteroidetes taxa discriminated human wastes, grazers, and pinnipeds from each other. We found 1058 different bacterial taxa that were unique to either human, grazing mammal, or bird fecal wastes. These OTUs can serve as specific identifier taxa for these sources in environmental waters. Two field tests in marine waters demonstrate the capacity of phylogenetic microarray analysis to track multiple sources with one test.

  16. New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

    PubMed

    Pérez-Amador, M A; Lidder, P; Johnson, M A; Landgraf, J; Wisman, E; Green, P J

    2001-12-01

    In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

  17. Integration of optical waveguides and microfluidics in a miniaturized antibody micro-array system for life detection in the NASA/ESA ExoMars mission

    NASA Astrophysics Data System (ADS)

    Prak, A.; Leeuwis, H.; Heideman, R. G.; Leinse, A.; Borst, G.

    2011-02-01

    Novel developments in antibody micro-array technology allow the development of very sensitive instrument that is capable of detecting a wide variety of different biomarkers from a sample liquid. An international consortium led by the UK is currently developing the Life Marker Chip as an analytical instrument for the ExoMars mission in 2018 based on the use of immunoassay technique. In this paper it will be discussed how micro/nano system hardware has been designed and the connected fabrication technology has been developed, compatible with the requirements of a Mars mission instrument and allowing a seamless integration in the instrument. A microfluidic fused silica chip integrates all the relevant components for the analysis/assay procedure (except the pumping, which is performed by a syringe-type bellows pump). The fluidic chip therefore contains an entries for intake of the pretreated sample, chambers for the solution of preloaded reagents and the hybridization reaction, liquid front sensors, inputs and output ports for the selector valve and a channel structure connecting these components. Moreover, the design has three parallel fluidic pathways in order to allow for three different classes of assays. The whole fluidic design is driven by the requirement that the dead volumes and the total liquid volume are as small as possible. It appeared that a miniaturized and integrated selector valve has far better properties than a system with numerous integrated and externally, often pneumatically actuated on-off valves. Next to this, the connected volume and mass of the whole fluid management system is lower. An optical array chip incorporates integrated waveguides, which allow for excitation of the fluorescent labels by the evanescent field of the guided light wave. The system had to be designed in such a way that the light of a single fibercoupled lightsource is distributed over all the spots (10 x 10) of the array. The LioniX proprietary waveguide technology Tri

  18. Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses

    PubMed Central

    Ge, Xinhui; Gebe, John A.; Bollyky, Paul L.; James, Eddie A.; Yang, Junbao; Stern, Lawrence J.; Kwok, William W.

    2010-01-01

    Background Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation. Methodology/Principal Findings We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells. Conclusions/Significance The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings. PMID:20634998

  19. 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

  20. DNA microarray analysis of the liver of mice treated with cobalt chloride.

    PubMed

    Matsumoto, Kanako; Fujishiro, Hitomi; Satoh, Masahiko; Himeno, Seiichiro

    2010-12-01

    To investigate the in vivo effects of cobalt chloride on gene expression at early time points, DNA microarray analysis was performed on the liver of mice injected subcutaneously with cobalt chloride. The liver tissue samples were taken 0.5, 1, and 3 hr after injection. Of the 14 genes up-regulated at 0.5 hr after injection, 7 are related to immunological responses, and 4 of the 7 were found to be involved in the activation of interferon.

  1. Clinical Presentation and Microarray Analysis of Peruvian Children with Atypical Development and/or Aberrant Behavior

    PubMed Central

    Butler, Merlin G.; Usrey, Kelly; Roberts, Jennifer L.; Schroeder, Stephen R.

    2014-01-01

    We report our experience with high resolution microarray analysis in infants and young children with developmental disability and/or aberrant behavior enrolled at the Centro Ann Sullivan del Peru in Lima, Peru, a low income country. Buccal cells were collected with cotton swabs from 233 participants for later DNA isolation and identification of copy number variation (deletions/duplications) and regions of homozygosity (ROH) for estimating consanguinity status in 15 infants and young children (12 males, 3 females; mean age ± SD = 28.1 m ±  7.9 m; age range 14 m–41 m) randomly selected for microarray analysis. An adequate DNA yield was found in about one-half of the enrolled participants. Ten participants showed deletions or duplications containing candidate genes reported to impact behavior or cognitive development. Five children had ROHs which could have harbored recessive gene alleles contributing to their clinical presentation. The coefficient of inbreeding was calculated and three participants showed first-second cousin relationships, indicating consanguinity. Our preliminary study showed that DNA isolated from buccal cells using cotton swabs was suboptimal, but yet in a subset of participants the yield was adequate for high resolution microarray analysis and several genes were found that impact development and behavior and ROHs identified to determine consanguinity status. PMID:25400949

  2. COMPARATIVE ANALYSIS OF MICROARRAY DATA IDENTIFIES COMMON RESPONSES TO CALORIC RESTRICTION AMONG MOUSE TISSUES

    PubMed Central

    Swindell, William R.

    2009-01-01

    Caloric restriction has been extensively investigated as an intervention that both extends lifespan and delays age-related disease in mammals. In mice, much interest has centered on evaluating gene expression changes induced by caloric restriction (CR) in particular tissue types, but the overall systemic effect of CR among multiple tissues has been examined less extensively. This study presents a comparative analysis of microarray datasets that have collectively examined the effects of CR in ten different tissue types (liver, heart, muscle, hypothalamus, hippocampus, white adipose tissue, colon, kidney, lung, cochlea). Using novel methods for comparative analysis of microarray data, detailed comparisons of the effects of CR among tissues are provided, and 28 genes for which expression response to CR is most shared among tissues are identified. These genes characterize common responses to CR, which consist of both activation and inhibition of stress-response pathways. With respect to liver tissue, transcriptional effects of CR exhibited surprisingly little overlap with those of aging, and a variable degree of overlap with the potential CR-mimetic drug resveratrol. These analyses shed light on the systemic transcriptional activity associated with CR diets, and also illustrate new approaches for comparative analysis of microarray datasets in the context of aging biology. PMID:18155270

  3. Microarray analysis reveals altered circulating microRNA expression in mice infected with Coxsackievirus B3

    PubMed Central

    Sun, Chaoyu; Tong, Lei; Zhao, Wenran; Wang, Yan; Meng, Yuan; Lin, Lexun; Liu, Bingchen; Zhai, Yujia; Zhong, Zhaohua; Li, Xueqi

    2016-01-01

    Coxsackievirus B3 (CVB3) is a common causative agent in the development of inflammatory cardiomyopathy. However, whether the expression of peripheral blood microRNAs (miRNAs) is altered in this process is unknown. The present study investigated changes to miRNA expression in the peripheral blood of CVB3-infected mice. Utilizing miRNA microarray technology, differential miRNA expression was examined between normal and CVB3-infected mice. The present results suggest that specific miRNAs were differentially expressed in the peripheral blood of mice infected with CVB3, varying with infection duration. Using miRNA microarray analysis, a total of 96 and 89 differentially expressed miRNAs were identified in the peripheral blood of mice infected with CVB3 for 3 and 6 days, respectively. Quantitative polymerase chain reaction was used to validate differentially expressed miRNAs, revealing a consistency of these results with the miRNA microarray analysis results. The biological functions of the differentially expressed miRNAs were then predicted by bioinformatics analysis. The potential biological roles of differentially expressed miRNAs included hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. These results may provide important insights into the mechanisms responsible for the progression of CVB3 infection. PMID:27698715

  4. Biofunctionalization of surfaces by energetic ion implantation: Review of progress on applications in implantable biomedical devices and antibody microarrays

    NASA Astrophysics Data System (ADS)

    Bilek, Marcela M. M.

    2014-08-01

    Despite major research efforts in the field of biomaterials, rejection, severe immune responses, scar tissue and poor integration continue to seriously limit the performance of today's implantable biomedical devices. Implantable biomaterials that interact with their host via an interfacial layer of active biomolecules to direct a desired cellular response to the implant would represent a major and much sought after improvement. Another, perhaps equally revolutionary, development that is on the biomedical horizon is the introduction of cost-effective microarrays for fast, highly multiplexed screening for biomarkers on cell membranes and in a variety of analyte solutions. Both of these advances will rely on effective methods of functionalizing surfaces with bioactive molecules. After a brief introduction to other methods currently available, this review will describe recently developed approaches that use energetic ions extracted from plasma to facilitate simple, one-step covalent surface immobilization of bioactive molecules. A kinetic theory model of the immobilization process by reactions with long-lived, mobile, surface-embedded radicals will be presented. The roles of surface chemistry and microstructure of the ion treated layer will be discussed. Early progress on applications of this technology to create diagnostic microarrays and to engineer bioactive surfaces for implantable biomedical devices will be reviewed.

  5. Outcome-Driven Cluster Analysis with Application to Microarray Data.

    PubMed

    Hsu, Jessie J; Finkelstein, Dianne M; Schoenfeld, David A

    2015-01-01

    One goal of cluster analysis is to sort characteristics into groups (clusters) so that those in the same group are more highly correlated to each other than they are to those in other groups. An example is the search for groups of genes whose expression of RNA is correlated in a population of patients. These genes would be of greater interest if their common level of RNA expression were additionally predictive of the clinical outcome. This issue arose in the context of a study of trauma patients on whom RNA samples were available. The question of interest was whether there were groups of genes that were behaving similarly, and whether each gene in the cluster would have a similar effect on who would recover. For this, we develop an algorithm to simultaneously assign characteristics (genes) into groups of highly correlated genes that have the same effect on the outcome (recovery). We propose a random effects model where the genes within each group (cluster) equal the sum of a random effect, specific to the observation and cluster, and an independent error term. The outcome variable is a linear combination of the random effects of each cluster. To fit the model, we implement a Markov chain Monte Carlo algorithm based on the likelihood of the observed data. We evaluate the effect of including outcome in the model through simulation studies and describe a strategy for prediction. These methods are applied to trauma data from the Inflammation and Host Response to Injury research program, revealing a clustering of the genes that are informed by the recovery outcome.

  6. Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes

    PubMed Central

    Tucker, Tracy; Zahir, Farah R; Griffith, Malachi; Delaney, Allen; Chai, David; Tsang, Erica; Lemyre, Emmanuelle; Dobrzeniecka, Sylvia; Marra, Marco; Eydoux, Patrice; Langlois, Sylvie; Hamdan, Fadi F; Michaud, Jacques L; Friedman, Jan M

    2014-01-01

    Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis. PMID:24253858

  7. Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes.

    PubMed

    Tucker, Tracy; Zahir, Farah R; Griffith, Malachi; Delaney, Allen; Chai, David; Tsang, Erica; Lemyre, Emmanuelle; Dobrzeniecka, Sylvia; Marra, Marco; Eydoux, Patrice; Langlois, Sylvie; Hamdan, Fadi F; Michaud, Jacques L; Friedman, Jan M

    2014-06-01

    Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis.

  8. Microarray analysis of differentially expressed genes engaged in fruit development between Prunus mume and Prunus armeniaca.

    PubMed

    Li, Xiaoying; Korir, Nicholas Kibet; Liu, Lili; Shangguan, Lingfei; Wang, Yuzhu; Han, Jian; Chen, Ming; Fang, Jinggui

    2012-11-15

    Microarray analysis is a technique that can be employed to provide expression profiles of single genes and new insights to elucidate the biological mechanisms responsible for fruit development. To evaluate expression of genes mostly engaged in fruit development between Prunus mume and Prunus armeniaca, we first identified differentially expressed transcripts along the entire fruit life cycle by using microarrays spotted with 10,641 ESTs collected from P. mume and other Prunus EST sequences. A total of 1418 ESTs were selected after quality control of microarray spots and analysis for differential gene expression patterns during fruit development of P. mume and P. Armeniaca. From these, 707 up-regulated and 711 down-regulated genes showing more than two-fold differences in expression level were annotated by GO based on biological processes, molecular functions and cellular components. These differentially expressed genes were found to be involved in several important pathways of carbohydrate, galactose, and starch and sucrose metabolism as well as in biosynthesis of other secondary metabolites via KEGG. This could provide detailed information on the fruit quality differences during development and ripening of these two species. With the results obtained, we provide a practical database for comprehensive understanding of molecular events during fruit development and also lay a theoretical foundation for the cloning of genes regulating in a series of important rate-limiting enzymes involved in vital metabolic pathways during fruit development.

  9. BayGO: Bayesian analysis of ontology term enrichment in microarray data

    PubMed Central

    Vêncio, Ricardo ZN; Koide, Tie; Gomes, Suely L; de B Pereira, Carlos A

    2006-01-01

    Background The search for enriched (aka over-represented or enhanced) ontology terms in a list of genes obtained from microarray experiments is becoming a standard procedure for a system-level analysis. This procedure tries to summarize the information focussing on classification designs such as Gene Ontology, KEGG pathways, and so on, instead of focussing on individual genes. Although it is well known in statistics that association and significance are distinct concepts, only the former approach has been used to deal with the ontology term enrichment problem. Results BayGO implements a Bayesian approach to search for enriched terms from microarray data. The R source-code is freely available at in three versions: Linux, which can be easily incorporated into pre-existent pipelines; Windows, to be controlled interactively; and as a web-tool. The software was validated using a bacterial heat shock response dataset, since this stress triggers known system-level responses. Conclusion The Bayesian model accounts for the fact that, eventually, not all the genes from a given category are observable in microarray data due to low intensity signal, quality filters, genes that were not spotted and so on. Moreover, BayGO allows one to measure the statistical association between generic ontology terms and differential expression, instead of working only with the common significance analysis. PMID:16504085

  10. Integration of microarray analysis into the clinical diagnosis of hematological malignancies: How much can we improve cytogenetic testing?

    PubMed Central

    Peterson, Jess F.; Aggarwal, Nidhi; Smith, Clayton A.; Gollin, Susanne M.; Surti, Urvashi; Rajkovic, Aleksandar; Swerdlow, Steven H.; Yatsenko, Svetlana A.

    2015-01-01

    Purpose To evaluate the clinical utility, diagnostic yield and rationale of integrating microarray analysis in the clinical diagnosis of hematological malignancies in comparison with classical chromosome karyotyping/fluorescence in situ hybridization (FISH). Methods G-banded chromosome analysis, FISH and microarray studies using customized CGH and CGH+SNP designs were performed on 27 samples from patients with hematological malignancies. A comprehensive comparison of the results obtained by three methods was conducted to evaluate benefits and limitations of these techniques for clinical diagnosis. Results Overall, 89.7% of chromosomal abnormalities identified by karyotyping/FISH studies were also detectable by microarray. Among 183 acquired copy number alterations (CNAs) identified by microarray, 94 were additional findings revealed in 14 cases (52%), and at least 30% of CNAs were in genomic regions of diagnostic/prognostic significance. Approximately 30% of novel alterations detected by microarray were >20 Mb in size. Balanced abnormalities were not detected by microarray; however, of the 19 apparently “balanced” rearrangements, 55% (6/11) of recurrent and 13% (1/8) of non-recurrent translocations had alterations at the breakpoints discovered by microarray. Conclusion Microarray technology enables accurate, cost-effective and time-efficient whole-genome analysis at a resolution significantly higher than that of conventional karyotyping and FISH. Array-CGH showed advantage in identification of cryptic imbalances and detection of clonal aberrations in population of non-dividing cancer cells and samples with poor chromosome morphology. The integration of microarray analysis into the cytogenetic diagnosis of hematologic malignancies has the potential to improve patient management by providing clinicians with additional disease specific and potentially clinically actionable genomic alterations. PMID:26299921

  11. Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibody array was developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli O serogroups. Sensitivity of the array was 10**5 CFU, and the limit of detection of serogroups in ground beef was 1-10 CFU following 12 h of enrichment. The array utilized a minimal amount...

  12. RL-SAGE and microarray analysis of the rice transcriptome after Rhizoctonia solani infection.

    PubMed

    Venu, R C; Jia, Yulin; Gowda, Malali; Jia, Melissa H; Jantasuriyarat, Chatchawan; Stahlberg, Eric; Li, Huameng; Rhineheart, Andrew; Boddhireddy, Prashanth; Singh, Pratibha; Rutger, Neil; Kudrna, David; Wing, Rod; Nelson, James C; Wang, Guo-Liang

    2007-10-01

    Sheath blight caused by the fungal pathogen Rhizoctonia solani is an emerging problem in rice production worldwide. To elucidate the molecular basis of rice defense to the pathogen, RNA isolated from R. solani-infected leaves of Jasmine 85 was used for both RL-SAGE library construction and microarray hybridization. RL-SAGE sequence analysis identified 20,233 and 24,049 distinct tags from the control and inoculated libraries, respectively. Nearly half of the significant tags (> or =2 copies) from both libraries matched TIGR annotated genes and KOME full-length cDNAs. Among them, 42% represented sense and 7% antisense transcripts, respectively. Interestingly, 60% of the library-specific (> or =10 copies) and differentially expressed (>4.0-fold change) tags were novel transcripts matching genomic sequence but not annotated genes. About 70% of the genes identified in the SAGE libraries showed similar expression patterns (up or down-regulated) in the microarray data obtained from three biological replications. Some candidate RL-SAGE tags and microarray genes were located in known sheath blight QTL regions. The expression of ten differentially expressed RL-SAGE tags was confirmed with RT-PCR. The defense genes associated with resistance to R. solani identified in this study are useful genomic materials for further elucidation of the molecular basis of the defense response to R. solani and fine mapping of target sheath blight QTLs.

  13. Global gene expression analysis of two Streptococcus thermophilus bacteriophages using DNA microarray.

    PubMed

    Duplessis, Martin; Russell, W Michael; Romero, Dennis A; Moineau, Sylvain

    2005-09-30

    A custom microarray was developed to study the temporal gene expression of the two groups of phages infecting the Gram-positive lactic acid bacterium Streptococcus thermophilus. The complete genomic sequence of the virulent cos-type phage DT1 (34,815 bp) and the pac-type phage 2972 (34,704 bp) were used for the construction of the microarray. Gene expression was measured at nine time intervals (0, 2, 7, 12, 17, 22, 27, 32 and 37 min) during phage infection and an expression curve was determined for each gene. Each phage gene was then classified into one of the three traditional transcription classes and these data were used to generate the complete transcriptional map of DT1 and 2972. Phage DT1 possesses 18 early genes, 12 middle genes and 12 late-expressed genes whereas 2972 has 16 early, 11 middle and 14 late genes. The trends of the phage gene expression profiles were also confirmed by slot blot hybridizations. Significant differences were observed when comparing the transcriptional maps of DT1 and 2972 with those already available for the S. thermophilus phages Sfi19 and Sfi21. To our knowledge, this report presents the first complete transcription analysis of bacteriophages infecting Gram-positive bacteria using the DNA microarray technology.

  14. Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays

    PubMed Central

    Gribble, Susan M; Ng, Bee Ling; Prigmore, Elena; Fitzgerald, Tomas; Carter, Nigel P

    2012-01-01

    Aarray painting is a technique that uses microarray technology to rapidly map chromosome translocation breakpoints. previous methods to map translocation breakpoints have used fluorescence in situ hybridization (FIsH) and have consequently been labor-intensive, time-consuming and restricted to the low breakpoint resolution imposed by the use of metaphase chromosomes. array painting combines the isolation of derivative chromosomes (chromosomes with translocations) and high-resolution microarray analysis to refine the genomic location of translocation breakpoints in a single experiment. In this protocol, we describe array painting by isolation of derivative chromosomes using a MoFlo flow sorter, amplification of these derivatives using whole-genome amplification and hybridization onto commercially available oligonucleotide microarrays. although the sorting of derivative chromosomes is a specialized procedure requiring sophisticated equipment, the amplification, labeling and hybridization of Dna is straightforward, robust and can be completed within 1 week. the protocol described produces good quality data; however, array painting is equally achievable using any combination of the available alternative methodologies for chromosome isolation, amplification and hybridization. PMID:19893508

  15. Neural network analysis of lymphoma microarray data: prognosis and diagnosis near-perfect

    PubMed Central

    O'Neill, Michael C; Song, Li

    2003-01-01

    Background Microarray chips are being rapidly deployed as a major tool in genomic research. To date most of the analysis of the enormous amount of information provided on these chips has relied on clustering techniques and other standard statistical procedures. These methods, particularly with regard to cancer patient prognosis, have generally been inadequate in providing the reduced gene subsets required for perfect classification. Results Networks trained on microarray data from DLBCL lymphoma patients have, for the first time, been able to predict the long-term survival of individual patients with 100% accuracy. Other networks were able to distinguish DLBCL lymphoma donors from other donors, including donors with other lymphomas, with 99% accuracy. Differentiating the trained network can narrow the gene profile to less than three dozen genes for each classification. Conclusions Here we show that artificial neural networks are a superior tool for digesting microarray data both with regard to making distinctions based on the data and with regard to providing very specific reference as to which genes were most important in making the correct distinction in each case. PMID:12697066

  16. Extreme value distribution based gene selection criteria for discriminant microarray data analysis using logistic regression.

    PubMed

    Li, Wentian; Sun, Fengzhu; Grosse, Ivo

    2004-01-01

    One important issue commonly encountered in the analysis of microarray data is to decide which and how many genes should be selected for further studies. For discriminant microarray data analyses based on statistical models, such as the logistic regression models, gene selection can be accomplished by a comparison of the maximum likelihood of the model given the real data, L(D|M), and the expected maximum likelihood of the model given an ensemble of surrogate data with randomly permuted label, L(D(0)|M). Typically, the computational burden for obtaining L(D(0)M) is immense, often exceeding the limits of available computing resources by orders of magnitude. Here, we propose an approach that circumvents such heavy computations by mapping the simulation problem to an extreme-value problem. We present the derivation of an asymptotic distribution of the extreme-value as well as its mean, median, and variance. Using this distribution, we propose two gene selection criteria, and we apply them to two microarray datasets and three classification tasks for illustration.

  17. Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

    PubMed

    Hot, D; Antoine, R; Renauld-Mongénie, G; Caro, V; Hennuy, B; Levillain, E; Huot, L; Wittmann, G; Poncet, D; Jacob-Dubuisson, F; Guyard, C; Rimlinger, F; Aujame, L; Godfroid, E; Guiso, N; Quentin-Millet, M-J; Lemoine, Y; Locht, C

    2003-07-01

    The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

  18. Subunit mass analysis for monitoring antibody oxidation

    PubMed Central

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping

    2017-01-01

    ABSTRACT Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. PMID:28106519

  19. Subunit mass analysis for monitoring antibody oxidation.

    PubMed

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  20. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

    PubMed Central

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  1. Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

    PubMed

    Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

    2012-10-16

    The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

  2. High-content single-cell analysis on-chip using a laser microarray scanner.

    PubMed

    Zhou, Jing; Wu, Yu; Lee, Sang-Kwon; Fan, Rong

    2012-12-07

    High-content cellomic analysis is a powerful tool for rapid screening of cellular responses to extracellular cues and examination of intracellular signal transduction pathways at the single-cell level. In conjunction with microfluidics technology that provides unique advantages in sample processing and precise control of fluid delivery, it holds great potential to transform lab-on-a-chip systems for high-throughput cellular analysis. However, high-content imaging instruments are expensive, sophisticated, and not readily accessible. Herein, we report on a laser scanning cytometry approach that exploits a bench-top microarray scanner as an end-point reader to perform rapid and automated fluorescence imaging of cells cultured on a chip. Using high-content imaging analysis algorithms, we demonstrated multiplexed measurements of morphometric and proteomic parameters from all single cells. Our approach shows the improvement of both sensitivity and dynamic range by two orders of magnitude as compared to conventional epifluorescence microscopy. We applied this technology to high-throughput analysis of mesenchymal stem cells on an extracellular matrix protein array and characterization of heterotypic cell populations. This work demonstrates the feasibility of a laser microarray scanner for high-content cellomic analysis and opens up new opportunities to conduct informative cellular analysis and cell-based screening in the lab-on-a-chip systems.

  3. Validation of a low-cost modified technique for constructing tissue microarrays for canine mammary tumor analysis.

    PubMed

    Silva, Franciele Basso Fernandes; Leite, Juliana da Silva; de Mello, Marcela Freire Vallim; Ferreira, Ana Maria Reis

    2016-09-01

    Compared with conventional histological paraffin blocks, tissue microarray (TMA) represents a "high-throughput tool" that provides rapid results, a time- and cost-effective approach and simultaneous investigation of several tissue samples under the same conditions. Given the large number of cases of dogs affected with mammary tumors, the complexity of these tumors and their similarity with breast cancer in women, this study aimed to validate a low-cost modified method to construct TMAs for canine mammary tumor analysis using immunomarkers. Carcinoma cases were selected from canine mammary carcinomas in mixed tumors (CMT) because this tumor type is the most heterogeneous among the histopathological types of mammary tumors observed in female dogs. Through a histopathological examination, tumor representativity was compared between conventional sections and histological sections obtained from the TMA block; both were stained with hematoxylin and eosin. An immunohistochemistry analysis was performed to compare the percentages of immunoreactive cells obtained in whole tissue sections versus those obtained from sections from the TMA block. Streptavidin-biotin peroxidase complex and anti-PCNA, anti-vimentin and anti-pancytokeratin antibodies were used. Statistical analysis consisted of the nonparametric Friedman's test (p≤0.05) and descriptive statistical analysis. Histopathological analysis showed tumor representativity in all TMA cores selected for the study. There was no difference between the immunohistochemical analysis of mammary tumors using conventional histological sections or sections obtained from a single 1-mm-diameter TMA core, regardless of the marker used: PCNA (p=0.279), pancytokeratin (p=0.243) and vimentin (p=0.967). The results did not change even when the means of any number of cores were compared among each other and with the conventional histological section: PCNA (p=0.413), pancytokeratin (p=0.177) and vimentin (p=1.0). Therefore, this study

  4. Analysis of microarray leukemia data using an efficient MapReduce-based K-nearest-neighbor classifier.

    PubMed

    Kumar, Mukesh; Rath, Nitish Kumar; Rath, Santanu Kumar

    2016-04-01

    Microarray-based gene expression profiling has emerged as an efficient technique for classification, prognosis, diagnosis, and treatment of cancer. Frequent changes in the behavior of this disease generates an enormous volume of data. Microarray data satisfies both the veracity and velocity properties of big data, as it keeps changing with time. Therefore, the analysis of microarray datasets in a small amount of time is essential. They often contain a large amount of expression, but only a fraction of it comprises genes that are significantly expressed. The precise identification of genes of interest that are responsible for causing cancer are imperative in microarray data analysis. Most existing schemes employ a two-phase process such as feature selection/extraction followed by classification. In this paper, various statistical methods (tests) based on MapReduce are proposed for selecting relevant features. After feature selection, a MapReduce-based K-nearest neighbor (mrKNN) classifier is also employed to classify microarray data. These algorithms are successfully implemented in a Hadoop framework. A comparative analysis is done on these MapReduce-based models using microarray datasets of various dimensions. From the obtained results, it is observed that these models consume much less execution time than conventional models in processing big data.

  5. Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.

    PubMed

    Yang, Dan-Hui; Barari, Mehrnoosh; Arif, Basil M; Krell, Peter J

    2007-08-01

    A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

  6. Aberrant Expression Profile of Long Noncoding RNA in Human Sinonasal Squamous Cell Carcinoma by Microarray Analysis

    PubMed Central

    Meng, Ling-zhao; Sun, Jing-wu; Yang, Fan

    2016-01-01

    Objectives. This study aimed to identify aberrantly expressed long noncoding RNAs (lncRNAs) profile of sinonasal squamous cell carcinoma (SSCC) and explore their potential functions. Methods. We investigated lncRNA and mRNA expression in SSCC and paired adjacent noncancerous tissues obtained from 6 patients with microarrays. Gene ontology (GO) analysis and pathway analysis were utilized to investigate the gene function. Gene signal-network and lncRNA-mRNA network were depicted. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to validate 5 lncRNAs in a second set of paired SSCC and adjacent noncancerous tissues obtained from 22 additional patients. Results. We identified significantly differentially expressed lncRNAs (n = 3146) and mRNAs (n = 2208) in SSCC relative to noncancerous tissues. The GO annotation indicated that there are some core gene products that may be attributed to the progress of SSCC. The pathway analysis identified many pathways associated with cancer. The results of lncRNA-mRNA network and gene signal-network implied some core lncRNAs/mRNAs might play important roles in SSCC pathogenesis. The results of qRT-PCR showed that all of the 5 lncRNAs were differentially expressed and consistent with the microarray results. Conclusion. Our study is the first screening and analysis of lncRNAs expression profile in SSCC and may offer new insights into pathogenesis of this disease. PMID:28044124

  7. Separate-channel analysis of two-channel microarrays: recovering inter-spot information

    PubMed Central

    2013-01-01

    Background Two-channel (or two-color) microarrays are cost-effective platforms for comparative analysis of gene expression. They are traditionally analysed in terms of the log-ratios (M-values) of the two channel intensities at each spot, but this analysis does not use all the information available in the separate channel observations. Mixed models have been proposed to analyse intensities from the two channels as separate observations, but such models can be complex to use and the gain in efficiency over the log-ratio analysis is difficult to quantify. Mixed models yield test statistics for the null distributions can be specified only approximately, and some approaches do not borrow strength between genes. Results This article reformulates the mixed model to clarify the relationship with the traditional log-ratio analysis, to facilitate information borrowing between genes, and to obtain an exact distributional theory for the resulting test statistics. The mixed model is transformed to operate on the M-values and A-values (average log-expression for each spot) instead of on the log-expression values. The log-ratio analysis is shown to ignore information contained in the A-values. The relative efficiency of the log-ratio analysis is shown to depend on the size of the intraspot correlation. A new separate channel analysis method is proposed that assumes a constant intra-spot correlation coefficient across all genes. This approach permits the mixed model to be transformed into an ordinary linear model, allowing the data analysis to use a well-understood empirical Bayes analysis pipeline for linear modeling of microarray data. This yields statistically powerful test statistics that have an exact distributional theory. The log-ratio, mixed model and common correlation methods are compared using three case studies. The results show that separate channel analyses that borrow strength between genes are more powerful than log-ratio analyses. The common correlation analysis

  8. 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.

  9. Diagnostic biomarkers for renal cell carcinoma: selection using novel bioinformatics systems for microarray data analysis

    PubMed Central

    Osunkoya, Adeboye O; Yin-Goen, Qiqin; Phan, John H; Moffitt, Richard A; Stokes, Todd H; Wang, May D; Young, Andrew N

    2009-01-01

    Summary The differential diagnosis of clear cell, papillary and chromophobe renal cell carcinoma is clinically important, because these tumor subtypes are associated with different pathobiology and clinical behavior. For cases in which histopathology is equivocal, immunohistochemistry and quantitative RT-PCR can assist in the differential diagnosis by measuring expression of subtype-specific biomarkers. Several renal tumor biomarkers have been discovered in expression microarray studies. However, due to heterogeneity of gene and protein expression, additional biomarkers are needed for reliable diagnostic classification. We developed novel bioinformatics systems to identify candidate renal tumor biomarkers from the microarray profiles of 45 clear cell, 16 papillary and 10 chromophobe renal cell carcinoma; the microarray data was derived from two independent published studies. The ArrayWiki biocomputing system merged the microarray datasets into a single file, so gene expression could be analyzed from a larger number of tumors. The caCORRECT system removed non-random sources of error from the microarray data, and the omniBioMarker system analyzed data with several gene-ranking algorithms, in order to identify algorithms effective at recognizing previously described renal tumor biomarkers. We predicted these algorithms would also be effective at identifying unknown biomarkers that could be verified by independent methods. We selected six novel candidate biomakers from the omniBioMarker analysis, and verified their differential expression in formalin-fixed paraffin-embedded tissues by quantitative RT-PCR and immunohistochemistry. The candidate biomarkers were carbonic anhydrase IX, ceruloplasmin, schwannomin-interacting protein 1, E74-like factor 3, cytochrome c oxidase subunit 5a and acetyl-CoA acetyltransferase 1. Quantitative RT-PCR was performed on 17 clear cell, 13 papillary and 7 chromophobe renal cell carcinoma. Carbonic anhydrase IX and ceruloplasmin were

  10. Microarray analysis of differentially expressed genes regulating lipid metabolism during melanoma progression.

    PubMed

    Sumantran, Venil N; Mishra, Pratik; Sudhakar, N

    2015-04-01

    A new hallmark of cancer involves acquisition of a lipogenic phenotype which promotes tumorigenesis. Little is known about lipid metabolism in melanomas. Therefore, we used BRB (Biometrics Research Branch) class comparison tool with multivariate analysis to identify differentially expressed genes in human cutaneous melanomas, compared with benign nevi and normal skin derived from the microarray dataset (GDS1375). The methods were validated by identifying known melanoma biomarkers (CITED1, FGFR2, PTPRF, LICAM, SPP1 and PHACTR1) in our results. Eighteen genes regulating metabolism of fatty acids, lipid second messengers and gangliosides were 2-9 fold upregulated in melanomas of GDS-1375. Out of the 18 genes, 13 were confirmed by KEGG pathway analysis and 10 were also significantly upregulated in human melanoma cell lines of NCI-60 Cell Miner database. Results showed that melanomas upregulated PPARGC1A transcription factor and its target genes regulating synthesis of fatty acids (SCD) and complex lipids (FABP3 and ACSL3). Melanoma also upregulated genes which prevented lipotoxicity (CPT2 and ACOT7) and regulated lipid second messengers, such as phosphatidic acid (AGPAT-4, PLD3) and inositol triphosphate (ITPKB, ITPR3). Genes for synthesis of pro-tumorigenic GM3 and GD3 gangliosides (UGCG, HEXA, ST3GAL5 and ST8SIA1) were also upregulated in melanoma. Overall, the microarray analysis of GDS-1375 dataset indicated that melanomas can become lipogenic by upregulating genes, leading to increase in fatty acid metabolism, metabolism of specific lipid second messengers, and ganglioside synthesis.

  11. Comprehensive Analysis of Prokaryotes in Environmental Water Using DNA Microarray Analysis and Whole Genome Amplification

    PubMed Central

    Akama, Takeshi; Kawashima, Akira; Tanigawa, Kazunari; Hayashi, Moyuru; Ishido, Yuko; Luo, Yuqian; Hata, Akihisa; Fujitani, Noboru; Ishii, Norihisa; Suzuki, Koichi

    2013-01-01

    The microflora in environmental water consists of a high density and diversity of bacterial species that form the foundation of the water ecosystem. Because the majority of these species cannot be cultured in vitro, a different approach is needed to identify prokaryotes in environmental water. A novel DNA microarray was developed as a simplified detection protocol. Multiple DNA probes were designed against each of the 97,927 sequences in the DNA Data Bank of Japan and mounted on a glass chip in duplicate. Evaluation of the microarray was performed using the DNA extracted from one liter of environmental water samples collected from seven sites in Japan. The extracted DNA was uniformly amplified using whole genome amplification (WGA), labeled with Cy3-conjugated 16S rRNA specific primers and hybridized to the microarray. The microarray successfully identified soil bacteria and environment-specific bacteria clusters. The DNA microarray described herein can be a useful tool in evaluating the diversity of prokaryotes and assessing environmental changes such as global warming. PMID:25437334

  12. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    PubMed

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

  13. A microarray analysis of gene expression patterns during early phases of newt lens regeneration

    PubMed Central

    Sousounis, Konstantinos; Michel, Christian S.; Bruckskotten, Marc; Maki, Nobuyasu; Borchardt, Thilo; Braun, Thomas; Tsonis, Panagiotis A.

    2013-01-01

    Purpose Notophthalmus viridescens, the red-spotted newt, possesses tremendous regenerative capabilities. Among the tissues and organs newts can regenerate, the lens is regenerated via transdifferentiation of the pigment epithelial cells of the dorsal iris, following complete removal (lentectomy). Under normal conditions, the same cells from the ventral iris are not capable of regenerating. This study aims to further understand the initial signals of lens regeneration. Methods We performed microarray analysis using RNA from a dorsal or ventral iris isolated 1, 3, and 5 days after lentectomy and compared to RNA isolated from an intact iris. This analysis was supported with quantitative real-time polymerase chain reaction (qRT-PCR) of selected genes. Results Microarrays showed 804 spots were differentially regulated 1, 3, and 5 days post-lentectomy in the dorsal and ventral iris. Functional annotation using Gene Ontology revealed interesting terms. Among them, factors related to cell cycle and DNA repair were mostly upregulated, in the microarray, 3 and 5 days post-lentectomy. qRT-PCR for rad1 and vascular endothelial growth factor receptor 1 showed upregulation for the dorsal iris 3 and 5 days post- lentectomy and for the ventral iris 5 days post-lentectomy. Rad1 was also upregulated twofold more in the dorsal iris than in the ventral iris 5 days post-lentectomy (p<0.001). Factors related to redox homeostasis were mostly upregulated in the microarray in all time points and samples. qRT-PCR for glutathione peroxidase 1 also showed upregulation in all time points for the ventral and dorsal iris. For the most part, mitochondrial enzymes were downregulated with the notable exception of cytochrome c–related oxidases that were mostly upregulated at all time points. qRT-PCR for cytochrome c oxidase subunit 2 showed upregulation especially 3 days post-lentectomy for the dorsal and ventral iris (p<0.001). Factors related to extracellular matrix and tissue remodeling showed

  14. Microarray Analysis of Human Liver Cells irradiated by 80MeV/u Carbon Ions

    NASA Astrophysics Data System (ADS)

    Wang, Xiao; Tian, Xiaoling; Kong, Fuquan; Li, Qiang; Jin, Xiaodong; Dai, Zhongying; Zhang, Hong; Yang, Mingjian; Zhao, Kui

    Objective Biological effect of heavy ion beam has the important significance for cancer therapy and space exploring owing its high LET and RBE, low OER, especially forming Bragg spike at the end of the tracks of charged particles. More serious damage for cells are induced by heavy ions and difficult repair than other irradiation such as X-ray and ν-ray . To explore the molecular mechanism of biological effect caused by heavy ionizing radiation (HIR) and to construct the gene expression profile database of HIR-induced human liver cells L02 by microarray analysis. Methods In this study, L02 cells were irradiated by 80MeV/u carbon ions at 5 Gy delivered by HIRFL (Heavy Ion Research Facility in Lanzhou) at room temperature. Total RNAs of cells incubated 6 hours and 24hours after irradiation were extracted with Trizol. Unirradiated cells were used as a control. RNAs were transcripted into cDNA by reverse transcription and labelled with cy5-dCTP and cy3-dCTP respectively. A human genome oligonucleotide set consisting of 5 amino acid-modified 70-mer probes and representing 21,329 well-characterized Homo sapiens genes was selected for microarray analysis and printed on amino-silaned glass slides. Arrays were fabricated using an OmniGrid microarrayer. Only genes whose alteration tendency was consistent in both microarrays were selected as differentially expressed genes. The Affymetrix's short oligonucleotide (25-mer) HG U133A 2.0 array analyses were performed per the manufacturer's instructions. Results Of the 21,329 genes tested, 37 genes showed changes in expression level with ratio higher than 2.0 and lower than 0.5 at 6hrs after irradiation. There were 19 genes showing up-regulation in radiated L02 cells, whereas 18 genes showing down-regulation; At 24hrs after irradiation, 269 genes showed changes in expression level with ratio higher than 2.0 and lower than 0.5. There were 67 genes showing up-regulation in radiated L02 cells, whereas 202 genes showing down

  15. Immune and inflammatory gene signature in rat cerebrum in subarachnoid hemorrhage with microarray analysis.

    PubMed

    Lee, Chu-I; Chou, An-Kuo; Lin, Ching-Chih; Chou, Chia-Hua; Loh, Joon-Khim; Lieu, Ann-Shung; Wang, Chih-Jen; Huang, Chi-Ying F; Howng, Shen-Long; Hong, Yi-Ren

    2012-01-01

    Cerebral vasospasm following subarachnoid hemorrhage (SAH) has been studied in terms of a contraction of the major cerebral arteries, but the effect of cerebrum tissue in SAH is not yet well understood. To gain insight into the biology of SAH-expressing cerebrum, we employed oligonucleotide microarrays to characterize the gene expression profiles of cerebrum tissue at the early stage of SAH. Functional gene expression in the cerebrum was analyzed 2 h following stage 1-hemorrhage in Sprague-Dawley rats. mRNA was investigated by performing microarray and quantitative real-time PCR analyses, and protein expression was determined by Western blot analysis. In this study, 18 upregulated and 18 downregulated genes displayed at least a 1.5-fold change. Five genes were verified by real-time PCR, including three upregulated genes [prostaglandin E synthase (PGES), CD14 antigen, and tissue inhibitor of metalloproteinase 1 (TIMP1)] as well as two downregulated genes [KRAB-zinc finger protein-2 (KZF-2) and γ-aminobutyric acid B receptor 1 (GABA B receptor)]. Notably, there were functional implications for the three upregulated genes involved in the inflammatory SAH process. However, the mechanisms leading to decreased KZF-2 and GABA B receptor expression in SAH have never been characterized. We conclude that oligonucleotide microarrays have the potential for use as a method to identify candidate genes associated with SAH and to provide novel investigational targets, including genes involved in the immune and inflammatory response. Furthermore, understanding the regulation of MMP9/TIMP1 during the early stages of SAH may elucidate the pathophysiological mechanisms in SAH rats.

  16. Loss of HITS (FAM107B) expression in cancers of multiple organs: tissue microarray analysis.

    PubMed

    Nakajima, Hideo; Koizumi, Keita; Tanaka, Takuji; Ishigaki, Yasuhito; Yoshitake, Yoshino; Yonekura, Hideto; Sakuma, Tsutomu; Fukushima, Toshihiro; Umehara, Hisanori; Ueno, Soichiro; Minamoto, Toshinari; Motoo, Yoshiharu

    2012-10-01

    Family with sequence similarity 107 (FAM107) proteins consist of two subtypes, FAM107A and FAM107B in mammals, possessing a conserved N-terminal domain of unknown function. Recently we found that FAM107B, an 18 kDa nuclear protein, is expressed in a broad range of tissues and is downregulated in gastrointestinal cancer. Because FAM107B expression is amplified by heat-shock stimulation, we designated it heat shock-inducible tumor small protein (HITS). Although data related to FAM107A as a candidate tumor suppressor have been accumulated, little biological information is available for HITS. In the present study, we examined HITS expression using immunohistochemistry with tissue microarrays and performed detailed statistical analyses. By screening a high-density multiple organ tumor and normal tissue microarray, HITS expression was decreased in tumor tissues of the breast, thyroid, testis and uterine cervix as well as the stomach and colon. Further analysis of tissue microarrays of individual organs showed that loss of HITS expression in cancer tissues was statistically significant and commonly observed in distinct organs in a histological type-specific manner. The HITS expression intensity was inversely correlated with the primary tumor size in breast and thyroid cancers. In addition, effects of tetracycline-inducible HITS expression on tumor growth were investigated in vivo. Forced expression of HITS inhibited tumor xenograft proliferation, compared with the mock-treated tumor xenograft model. These results show that loss of HITS expression is a common phenomenon observed in cancers of distinct organs and involved in tumor development and proliferation.

  17. Identification of Iron Homeostasis Genes Dysregulation Potentially Involved in Retinopathy of Prematurity Pathogenicity by Microarray Analysis

    PubMed Central

    Luo, Xian-qiong; Zhang, Chun-yi; Zhang, Jia-wen; Jiang, Jing-bo; Yin, Ai-hua; Guo, Li; Nie, Chuan; Lu, Xu-zai; Deng, Hua; Zhang, Liang

    2015-01-01

    Retinopathy of prematurity (ROP) is a serious disease of preterm neonates and there are limited systematic studies of the molecular mechanisms underlying ROP. Therefore, here we performed global gene expression profiling in human fetal retinal microvascular endothelial cells (RMECs) under hypoxic conditions in vitro. Aborted fetuses were enrolled and primary RMECs were isolated from eyeballs. Cultivated cells were treated with CoCl2 to induce hypoxia. The dual-color microarray approach was adopted to compare gene expression profiling between treated RMECs and the paired untreated control. The one-class algorithm in significance analysis of microarray (SAM) software was used to screen the differentially expressed genes (DEGs) and quantitative RT-PCR (qRT-PCR) was conducted to validate the results. Gene Ontology was employed for functional enrichment analysis. There were 326 DEGs between the hypoxia-induced group and untreated group. Of these genes, 198 were upregulated in hypoxic RMECs, while the other 128 hits were downregulated. In particular, genes in the iron ion homeostasis pathway were highly enriched under hypoxic conditions. Our study indicates that dysregulation of genes involved in iron homeostasis mediating oxidative damage may be responsible for the mechanisms underlying ROP. The “oxygen plus iron” hypothesis may improve our understanding of ROP pathogenesis. PMID:26557385

  18. Microarray and functional analysis of growth phase-dependent gene regulation in Bordetella bronchiseptica.

    PubMed

    Nicholson, Tracy L; Buboltz, Anne M; Harvill, Eric T; Brockmeier, Susan L

    2009-10-01

    Growth phase-dependent gene regulation has recently been demonstrated to occur in Bordetella pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived from a Bordetella bronchiseptica-like ancestor, we hypothesized that growth phase-dependent gene regulation would also occur in B. bronchiseptica. Microarray analysis revealed and quantitative real-time PCR (qRT-PCR) confirmed that growth phase-dependent gene regulation occurs in B. bronchiseptica, resulting in prominent temporal shifts in global gene expression. Two virulence phenotypes associated with these gene expression changes were tested. We found that growth-dependent increases in expression of some type III secretion system (TTSS) genes led to a growth phase-dependent increase in a TTSS-dependent function, cytotoxicity. Although the transcription of genes encoding adhesins previously shown to mediate adherence was decreased in late-log and stationary phases, we found that the adherence of B. bronchiseptica did not decrease in these later phases of growth. Microarray analysis revealed and qRT-PCR confirmed that growth phase-dependent gene regulation occurred in both Bvg(+) and Bvg(-) phase-locked mutants, indicating that growth phase-dependent gene regulation in B. bronchiseptica can function independently from the BvgAS regulatory system.

  19. Focused Microarray Analysis of Peripheral Mononuclear Blood Cells from Churg–Strauss Syndrome Patients

    PubMed Central

    Tougan, Takahiro; Onda, Hiroaki; Okuzaki, Daisuke; Kobayashi, Shigeto; Hashimoto, Hiroshi; Nojima, Hiroshi

    2008-01-01

    DNA diagnostics are useful but are hampered by difficult ethical issues. Moreover, it cannot provide enough information on the environmental factors that are important for pathogenesis of certain diseases. However, this is not a problem for RNA diagnostics, which evaluate the expression of the gene in question. We here report a novel RNA diagnostics tool that can be employed with peripheral blood mononuclear cells (PBMCs). To establish this tool, we identified 290 genes that are highly expressed in normal PBMCs but not in TIG-1, a normal human fibroblast cell. These genes were entitled PREP after predominantly expressed in PBMC and included 50 uncharacterized genes. We then conducted PREP gene-focused microarray analysis on PBMCs from seven cases of Churg–Strauss syndrome (CSS), which is a small-vessel necrotizing vasculitis. We found that PREP135 (coactosin-like protein), PREP77 (prosaposin), PREP191 (cathepsin D), PREP234 (c-fgr), and PREP136 (lysozyme) were very highly up-regulated in all seven CSS patients. Another 28 genes were also up-regulated, albeit more moderately, and three were down-regulated in all CSS patients. The nature of these up- and down-regulated genes suggest that the immune systems of the patients are activated in response to invading microorganisms. These observations indicate that focused microarray analysis of PBMCs may be a practical, useful, and low-cost bedside diagnostics tool. PMID:18263571

  20. DATE analysis: A general theory of biological change applied to microarray data.

    PubMed

    Rasnick, David

    2009-01-01

    In contrast to conventional data mining, which searches for specific subsets of genes (extensive variables) to correlate with specific phenotypes, DATE analysis correlates intensive state variables calculated from the same datasets. At the heart of DATE analysis are two biological equations of state not dependent on genetic pathways. This result distinguishes DATE analysis from other bioinformatics approaches. The dimensionless state variable F quantifies the relative overall cellular activity of test cells compared to well-chosen reference cells. The variable pi(i) is the fold-change in the expression of the ith gene of test cells relative to reference. It is the fraction phi of the genome undergoing differential expression-not the magnitude pi-that controls biological change. The state variable phi is equivalent to the control strength of metabolic control analysis. For tractability, DATE analysis assumes a linear system of enzyme-connected networks and exploits the small average contribution of each cellular component. This approach was validated by reproducible values of the state variables F, RNA index, and phi calculated from random subsets of transcript microarray data. Using published microarray data, F, RNA index, and phi were correlated with: (1) the blood-feeding cycle of the malaria parasite, (2) embryonic development of the fruit fly, (3) temperature adaptation of Killifish, (4) exponential growth of cultured S. pneumoniae, and (5) human cancers. DATE analysis was applied to aCGH data from the great apes. A good example of the power of DATE analysis is its application to genomically unstable cancers, which have been refractory to data mining strategies.

  1. Prediction of recurrence of non muscle-invasive bladder cancer by means of a protein signature identified by antibody microarray analyses.

    PubMed

    Srinivasan, Harish; Allory, Yves; Sill, Martin; Vordos, Dimitri; Alhamdani, Mohamed Saiel Saeed; Radvanyi, Francois; Hoheisel, Jörg D; Schröder, Christoph

    2014-06-01

    About 70% of newly diagnosed cases of bladder cancer are low-stage, low-grade, non muscle-invasive. Standard treatment is transurethral resection. About 60% of the tumors will recur, however, and in part progress to become invasive. Therefore, surveillance cystoscopy is performed after resection. However, in the USA and Europe alone, about 54 000 new patients per year undergo repeated cystoscopies over several years, who do not experience recurrence. Analysing in a pilot study resected tumors from patients with (n = 19) and without local recurrence (n = 6) after a period of 5 years by means of an antibody microarray that targeted 724 cancer-related proteins, we identified 255 proteins with significantly differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. A multivariate classifier was constructed based on 20 proteins. It facilitates the prediction of recurrence with a sensitivity of 80% and a specificity of 100%. As a measure of overall accuracy, the area under the curve value was found to be 91%. After validation in additional sample cohorts with a similarly long follow-up, such a signature could support decision making about the stringency of surveillance or even different treatment options.

  2. Pathway-based analysis of microarray and RNAseq data using Pathway Processor 2.0.

    PubMed

    Beltrame, Luca; Bianco, Luca; Fontana, Paolo; Cavalieri, Duccio

    2013-03-01

    The constant improvement of high-throughput technologies has led to a great increase in generated data per single experiment. Pathway analysis is a widespread method to understand experimental results at the system level. Pathway Processor 2.0 is an upgrade over the original Pathway Processor program developed in 2002, extended to support more species, analysis methods, and RNAseq data in addition to microarrays through a simple Web-based interface. The tool can perform two different types of analysis: the first covers the traditional Fisher's Test used by Pathway Processor and topology-aware analyses, which take into account the propagation of changes over the whole structure of a pathway, and the second is a new pathway-based method to investigate differences between phenotypes of interest. Common problems and troubleshooting are also discussed.

  3. Microarray Based Gene Expression Analysis of Murine Brown and Subcutaneous Adipose Tissue: Significance with Human

    PubMed Central

    Boparai, Ravneet K.; Kondepudi, Kanthi Kiran; Mantri, Shrikant; Bishnoi, Mahendra

    2015-01-01

    Background Two types of adipose tissues, white (WAT) and brown (BAT) are found in mammals. Increasingly novel strategies are being proposed for the treatment of obesity and its associated complications by altering amount and/or activity of BAT using mouse models. Methodology/Principle Findings The present study was designed to: (a) investigate the differential expression of genes in LACA mice subcutaneous WAT (sWAT) and BAT using mouse DNA microarray, (b) to compare mouse differential gene expression with previously published human data; to understand any inter- species differences between the two and (c) to make a comparative assessment with C57BL/6 mouse strain. In mouse microarray studies, over 7003, 1176 and 401 probe sets showed more than two-fold, five-fold and ten-fold change respectively in differential expression between murine BAT and WAT. Microarray data was validated using quantitative RT-PCR of key genes showing high expression in BAT (Fabp3, Ucp1, Slc27a1) and sWAT (Ms4a1, H2-Ob, Bank1) or showing relatively low expression in BAT (Pgk1, Cox6b1) and sWAT (Slc20a1, Cd74). Multi-omic pathway analysis was employed to understand possible links between the organisms. When murine two fold data was compared with published human BAT and sWAT data, 90 genes showed parallel differential expression in both mouse and human. Out of these 90 genes, 46 showed same pattern of differential expression whereas the pattern was opposite for the remaining 44 genes. Based on our microarray results and its comparison with human data, we were able to identify genes (targets) (a) which can be studied in mouse model systems to extrapolate results to human (b) where caution should be exercised before extrapolation of murine data to human. Conclusion Our study provides evidence for inter species (mouse vs human) differences in differential gene expression between sWAT and BAT. Critical understanding of this data may help in development of novel ways to engineer one form of adipose

  4. SegMine workflows for semantic microarray data analysis in Orange4WS

    PubMed Central

    2011-01-01

    Background In experimental data analysis, bioinformatics researchers increasingly rely on tools that enable the composition and reuse of scientific workflows. The utility of current bioinformatics workflow environments can be significantly increased by offering advanced data mining services as workflow components. Such services can support, for instance, knowledge discovery from diverse distributed data and knowledge sources (such as GO, KEGG, PubMed, and experimental databases). Specifically, cutting-edge data analysis approaches, such as semantic data mining, link discovery, and visualization, have not yet been made available to researchers investigating complex biological datasets. Results We present a new methodology, SegMine, for semantic analysis of microarray data by exploiting general biological knowledge, and a new workflow environment, Orange4WS, with integrated support for web services in which the SegMine methodology is implemented. The SegMine methodology consists of two main steps. First, the semantic subgroup discovery algorithm is used to construct elaborate rules that identify enriched gene sets. Then, a link discovery service is used for the creation and visualization of new biological hypotheses. The utility of SegMine, implemented as a set of workflows in Orange4WS, is demonstrated in two microarray data analysis applications. In the analysis of senescence in human stem cells, the use of SegMine resulted in three novel research hypotheses that could improve understanding of the underlying mechanisms of senescence and identification of candidate marker genes. Conclusions Compared to the available data analysis systems, SegMine offers improved hypothesis generation and data interpretation for bioinformatics in an easy-to-use integrated workflow environment. PMID:22029475

  5. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27941670

  7. Analysis of factorial time-course microarrays with application to a clinical study of burn injury.

    PubMed

    Zhou, Baiyu; Xu, Weihong; Herndon, David; Tompkins, Ronald; Davis, Ronald; Xiao, Wenzhong; Wong, Wing Hung; Toner, Mehmet; Warren, H Shaw; Schoenfeld, David A; Rahme, Laurence; McDonald-Smith, Grace P; Hayden, Douglas; Mason, Philip; Fagan, Shawn; Yu, Yong-Ming; Cobb, J Perren; Remick, Daniel G; Mannick, John A; Lederer, James A; Gamelli, Richard L; Silver, Geoffrey M; West, Michael A; Shapiro, Michael B; Smith, Richard; Camp, David G; Qian, Weijun; Storey, John; Mindrinos, Michael; Tibshirani, Rob; Lowry, Stephen; Calvano, Steven; Chaudry, Irshad; West, Michael A; Cohen, Mitchell; Moore, Ernest E; Johnson, Jeffrey; Moldawer, Lyle L; Baker, Henry V; Efron, Philip A; Balis, Ulysses G J; Billiar, Timothy R; Ochoa, Juan B; Sperry, Jason L; Miller-Graziano, Carol L; De, Asit K; Bankey, Paul E; Finnerty, Celeste C; Jeschke, Marc G; Minei, Joseph P; Arnoldo, Brett D; Hunt, John L; Horton, Jureta; Cobb, J Perren; Brownstein, Bernard; Freeman, Bradley; Maier, Ronald V; Nathens, Avery B; Cuschieri, Joseph; Gibran, Nicole; Klein, Matthew; O'Keefe, Grant

    2010-06-01

    Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It is important to study how these dynamic profiles depend on the multiple factors that characterize the experimental condition under which the time course is observed. Analytic methods are needed to simultaneously handle the time course and factorial structure in the data. We developed a method to evaluate factor effects by pooling information across the time course while accounting for multiple testing and nonnormality of the microarray data. The method effectively extracts gene-specific response features and models their dependency on the experimental factors. Both longitudinal and cross-sectional time-course data can be handled by our approach. The method was used to analyze the impact of age on the temporal gene response to burn injury in a large-scale clinical study. Our analysis reveals that 21% of the genes responsive to burn are age-specific, among which expressions of mitochondria and immunoglobulin genes are differentially perturbed in pediatric and adult patients by burn injury. These new findings in the body's response to burn injury between children and adults support further investigations of therapeutic options targeting specific age groups. The methodology proposed here has been implemented in R package "TANOVA" and submitted to the Comprehensive R Archive Network at http://www.r-project.org/. It is also available for download at http://gluegrant1.stanford.edu/TANOVA/.

  8. Functional analysis of differentially expressed genes associated with glaucoma from DNA microarray data.

    PubMed

    Wu, Y; Zang, W D; Jiang, W

    2014-11-11

    Microarray data of astrocytes extracted from the optic nerves of donors with and without glaucoma were analyzed to screen for differentially expressed genes (DEGs). Functional exploration with bioinformatic tools was then used to understand the roles of the identified DEGs in glaucoma. Microarray data were downloaded from the Gene Expression Omnibus (GEO) database, which contains 13 astrocyte samples, 6 from healthy subjects and 7 from patients suffering from glaucoma. Data were pre-processed, and DEGs were screened out using R software packages. Interactions between DEGs were identified, and networks were built using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). GENECODIS was utilized for the functional analysis of the DEGs, and GOTM was used for module division, for which functional annotation was conducted with the Database for Annotation, Visualization, and Integrated Discovery (DAVID). A total of 371 DEGs were identified between glaucoma-associated samples and normal samples. Three modules included in the PPID database were generated with 11, 12, and 2 significant functional annotations, including immune system processes, inflammatory responses, and synaptic vesicle endocytosis, respectively. We found that the most significantly enriched functions for each module were associated with immune function. Several genes that play interesting roles in the development of glaucoma are described; these genes may be potential biomarkers for glaucoma diagnosis or treatment.

  9. Application of the Taguchi method to the analysis of the deposition step in microarray production.

    PubMed

    Severgnini, Marco; Pattini, Linda; Consolandi, Clarissa; Rizzi, Ermanno; Battaglia, Cristina; De Bellis, Gianluca; Cerutti, Sergio

    2006-09-01

    Every microarray experiment is affected by many possible sources of variability that may even corrupt biological evidence on analyzed sequences. We applied a "Taguchi method" strategy, based on the use of orthogonal arrays to optimize the deposition step of oligonucleotide sequences on glass slides. We chose three critical deposition parameters (humidity, surface, and buffer) at two levels each, in order to establish optimum settings. A L8 orthogonal array was used in order to monitor both the main effects and interactions on the deposition of a 25 mer oligonucleotide hybridized to its fluorescent-labeled complementary. Signal-background ratio and deposition homogeneity in terms of mean intensity and spot diameter were considered as significant outputs. An analysis of variance (ANOVA) was applied to raw data and to mean results for each slide and experimental run. Finally we calculated an overall evaluation coefficient to group together important outputs in one number. Environmental humidity and surface-buffer interaction were recognized as the most critical factors, for which a 50% humidity, associated to a chitosan-covered slide and a sodium phosphate + 25% dimethyl sulfoxide (DMSO) buffer gave best performances. Our results also suggested that Taguchi methods can be efficiently applied in optimization of microarray procedures.

  10. Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.

    PubMed

    Gomes, Luciana I; Silva, Ricardo L A; Stolf, Beatriz S; Cristo, Elier B; Hirata, Roberto; Soares, Fernando A; Reis, Luiz F L; Neves, E Jordão; Carvalho, Alex F

    2003-10-15

    Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.

  11. Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray

    PubMed Central

    2012-01-01

    Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process. PMID:23256600

  12. cDNA Microarray Analysis of Serially Sampled Cervical Cancer Specimens From Patients Treated With Thermochemoradiotherapy

    SciTech Connect

    Borkamo, Erling Dahl; Schem, Baard-Christian; Fluge, Oystein; Bruland, Ove; Dahl, Olav; Mella, Olav

    2009-12-01

    Purpose: To elucidate changes in gene expression after treatment with regional thermochemoradiotherapy in locally advanced squamous cell cervical cancer. Methods and Materials: Tru-Cut biopsy specimens were serially collected from 16 patients. Microarray gene expression levels before and 24 h after the first and second trimodality treatment sessions were compared. Pathway and network analyses were conducted by use of Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA). Single gene expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. Results: We detected 53 annotated genes that were differentially expressed after trimodality treatment. Central in the three top networks detected by IPA were interferon alfa, interferon beta, and interferon gamma receptor; nuclear factor kappaB; and tumor necrosis factor, respectively. These genes encode proteins that are important in regulation cell signaling, proliferation, gene expression, and immune stimulation. Biological processes over-represented among the 53 genes were fibrosis, tumorigenesis, and immune response. Conclusions: Microarrays showed minor changes in gene expression after thermochemoradiotherapy in locally advanced cervical cancer. We detected 53 differentially expressed genes, mainly involved in fibrosis, tumorigenesis, and immune response. A limitation with the use of serial biopsy specimens was low quality of ribonucleic acid from tumors that respond to highly effective therapy. Another 'key limitation' is timing of the post-treatment biopsy, because 24 h may be too late to adequately assess the impact of hyperthermia on gene expression.

  13. Analysing breast cancer microarrays from African Americans using shrinkage-based discriminant analysis.

    PubMed

    Pang, Herbert; Ebisu, Keita; Watanabe, Emi; Sue, Laura Y; Tong, Tiejun

    2010-10-01

    Breast cancer tumours among African Americans are usually more aggressive than those found in Caucasian populations. African-American patients with breast cancer also have higher mortality rates than Caucasian women. A better understanding of the disease aetiology of these breast cancers can help to improve and develop new methods for cancer prevention, diagnosis and treatment. The main goal of this project was to identify genes that help differentiate between oestrogen receptor-positive and -negative samples among a small group of African-American patients with breast cancer. Breast cancer microarrays from one of the largest genomic consortiums were analysed using 13 African-American and 201 Caucasian samples with oestrogen receptor status. We used a shrinkage-based classification method to identify genes that were informative in discriminating between oestrogen receptor-positive and -negative samples. Subset analysis and permutation were performed to obtain a set of genes unique to the African-American population. We identified a set of 156 probe sets, which gave a misclassification rate of 0.16 in distinguishing between oestrogen receptor-positive and -negative patients. The biological relevance of our findings was explored through literature-mining techniques and pathway mapping. An independent dataset was used to validate our findings and we found that the top ten genes mapped onto this dataset gave a misclassification rate of 0.15. The described method allows us best to utilise the information available from small sample size microarray data in the context of ethnic minorities.

  14. Transcriptional Profiling of Hydrogen Production Metabolism of Rhodobacter capsulatus under Temperature Stress by Microarray Analysis

    PubMed Central

    Gürgan, Muazzez; Afşar Erkal, Nilüfer; Özgür, Ebru; Gündüz, Ufuk; Eroglu, Inci; Yücel, Meral

    2015-01-01

    Biohydrogen is a clean and renewable form of hydrogen, which can be produced by photosynthetic bacteria in outdoor large-scale photobioreactors using sunlight. In this study, the transcriptional response of Rhodobacter capsulatus to cold (4 °C) and heat (42 °C) stress was studied using microarrays. Bacteria were grown in 30/2 acetate/glutamate medium at 30 °C for 48 h under continuous illumination. Then, cold and heat stresses were applied for two and six hours. Growth and hydrogen production were impaired under both stress conditions. Microarray chips for R. capsulatus were custom designed by Affymetrix (GeneChip®. TR_RCH2a520699F). The numbers of significantly changed genes were 328 and 293 out of 3685 genes under cold and heat stress, respectively. Our results indicate that temperature stress greatly affects the hydrogen production metabolisms of R. capsulatus. Specifically, the expression of genes that participate in nitrogen metabolism, photosynthesis and the electron transport system were induced by cold stress, while decreased by heat stress. Heat stress also resulted in down regulation of genes related to cell envelope, transporter and binding proteins. Transcriptome analysis and physiological results were consistent with each other. The results presented here may aid clarification of the genetic mechanisms for hydrogen production in purple non-sulfur (PNS) bacteria under temperature stress. PMID:26086826

  15. DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish.

    PubMed

    Feroudj, Holger; Matsumoto, Takuya; Kurosu, Yohei; Kaneko, Gen; Ushio, Hideki; Suzuki, Katsuaki; Kondo, Hidehiro; Hirono, Ikuo; Nagashima, Yuji; Akimoto, Seiji; Usui, Kazushige; Kinoshita, Shigeharu; Asakawa, Shuichi; Kodama, Masaaki; Watabe, Shugo

    2014-01-01

    Pufferfish accumulate tetrodotoxin (TTX) at high levels in liver and ovary through the food chain. However, the mechanisms underlying TTX toxification in pufferfish have been poorly understood. In order to search gene candidates involved in TTX accumulation in the torafugu pufferfish Takifugu rubripes, a custom 4x44k oligonucleotide microarray slide was designed by the Agilent eArray program using oligonucleotide probes of 60 bp in length referring to 42,724 predicted transcripts in the publicly available Fugu genome database. DNA microarray analysis was performed with total RNA samples from the livers of two toxic wild specimens in comparison with those from a nontoxic wild specimen and two nontoxic cultured specimens. The mRNA levels of 1108 transcripts were more than 2-fold higher in the toxic specimens than in the nontoxic specimens. The levels of 613 transcripts were remarkably high, and 16 transcripts encoded by 9 genes were up-regulated more than 10-fold. These genes included those encoding forming structural filaments (keratins) and those related to vitamin D metabolism and immunity. It was also noted that the levels of the transcripts encoding serpin peptidase inhibitor clade C member 1, coagulation factor X precursor, complement C2, C3, C5, C8 precursors, and interleukin-6 receptor were high in the toxic liver samples.

  16. 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

  17. Microarray analysis of Neosartorya fischeri using different carbon sources, petroleum asphaltenes and glucose-peptone

    PubMed Central

    Hernández-López, Edna L.; Ramírez-Puebla, Shamayim T.; Vazquez-Duhalt, Rafael

    2015-01-01

    Asphaltenes are considered as the most recalcitrant petroleum fraction and represent a big problem for the recovery, separation and processing of heavy oils and bitumens. Neosartorya fischeri is a saprophytic fungus that is able to grow using asphaltenes as the sole carbon source [1]. We performed transcription profiling using a custom designed microarray with the complete genome from N. fischeri NRRL 181 in order to identify genes related to the transformation of asphaltenes [1]. Data analysis was performed using the genArise software. Results showed that 287 genes were up-regulated and 118 were down-regulated. Here we describe experimental procedures and methods about our dataset (NCBI GEO accession number GSE68146) and describe the data analysis to identify different expression levels in N. fischeri using this recalcitrant carbon source. PMID:26484261

  18. Microarray analysis of Neosartorya fischeri using different carbon sources, petroleum asphaltenes and glucose-peptone.

    PubMed

    Hernández-López, Edna L; Ramírez-Puebla, Shamayim T; Vazquez-Duhalt, Rafael

    2015-09-01

    Asphaltenes are considered as the most recalcitrant petroleum fraction and represent a big problem for the recovery, separation and processing of heavy oils and bitumens. Neosartorya fischeri is a saprophytic fungus that is able to grow using asphaltenes as the sole carbon source [1]. We performed transcription profiling using a custom designed microarray with the complete genome from N. fischeri NRRL 181 in order to identify genes related to the transformation of asphaltenes [1]. Data analysis was performed using the genArise software. Results showed that 287 genes were up-regulated and 118 were down-regulated. Here we describe experimental procedures and methods about our dataset (NCBI GEO accession number GSE68146) and describe the data analysis to identify different expression levels in N. fischeri using this recalcitrant carbon source.

  19. Administered chrysanthemum flower oil attenuates hyperuricemia: mechanism of action as revealed by DNA microarray analysis.

    PubMed

    Honda, Shinichi; Kawamoto, Seiji; Tanaka, Hozumi; Kishida, Hideyuki; Kitagawa, Masayasu; Nakai, Yuji; Abe, Keiko; Hirata, Dai

    2014-01-01

    We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.

  20. Chromosomal microarray analysis in a girl with mental retardation and spina bifida.

    PubMed

    Ben Abdallah, Inesse; Hannachi, Hanene; Soyah, Najla; Saad, Ali; Elghezal, Hatem

    2011-01-01

    Chromosomal imbalances comprise a major cause of mental retardation, particularly in association with congenital malformations and dysmorphic features. Chromosomal analysis using banded karyotyping is limited by the low resolution of this technique, and cryptic chromosomal rearrangements cannot be detected. We describe a 6-year-old girl with mental retardation, mild growth, congenital malformation, and facial anomalies. Chromosomal analysis with karyotyping produced normal results. Because the phenotype suggested chromosomal abnormality, microarray comparative genomic hybridization was used to search for a possible cryptic anomaly. A subtelomeric chromosomal imbalance, consisting of partial trisomy 2q35 and partial monosomy 3p26, was detected and confirmed using fluorescence in situ hybridization. This rearrangement was inherited from an equilibrated maternal t(2;3) reciprocal translocation. Comparative genomic hybridization array in similar situations is useful in detecting cryptic chromosomal rearrangements, identifying genes contained in deleted or duplicated regions, establishing a precise phenotype-genotype correlation, and offering unambiguous genetic counseling.

  1. Phytoremediation potential of Arabidopsis with reference to acrylamide and microarray analysis of acrylamide-response genes.

    PubMed

    Gao, Jian-Jie; Peng, Ri-He; Zhu, Bo; Wang, Bo; Wang, Li-Juan; Xu, Jing; Sun, Miao; Yao, Quan-Hong

    2015-10-01

    Acrylamide (ACR) is a widely used industrial chemical. However, it is a dangerous compound because it showed neurotoxic effects in humans and act as reproductive toxicant and carcinogen in many animal species. In the environment, acrylamide has high soil mobility and may travel via groundwater. Phytoremediation is an effective method to remove the environmental pollutants, but the mechanism of plant response to acrylamide remains unknown. With the purpose of assessing remediation potentials of plants for acrylamide, we have examined acrylamide uptake by the model plant Arabidopsis grown on contaminated substrates with high performance liquid chromatography (HPLC) analysis. The result revealed that acrylamide could be absorbed and degraded by Arabidopsis. Further microarray analysis showed that 527 transcripts were up-regulated within 2-days under acrylamide exposure condition. We have found many potential acrylamide-induced genes playing a major role in plant metabolism and phytoremediation.

  2. Adaptation of a Bioinformatics Microarray Analysis Workflow for a Toxicogenomic Study in Rainbow Trout

    PubMed Central

    Depiereux, Sophie; De Meulder, Bertrand; Bareke, Eric; Berger, Fabrice; Le Gac, Florence; Depiereux, Eric; Kestemont, Patrick

    2015-01-01

    Sex steroids play a key role in triggering sex differentiation in fish, the use of exogenous hormone treatment leading to partial or complete sex reversal. This phenomenon has attracted attention since the discovery that even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies on statistical analysis, species genomic resources, and bioinformatics tools. The goals of this study are to improve the knowledge of feminisation in fish, by the analysis of molecular responses in the gonads of rainbow trout fry after chronic exposure to several doses (0.01, 0.1, 1 and 10 μg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. We successfully adapted a bioinformatics microarray analysis workflow elaborated on human data to a toxicogenomic study using rainbow trout, a fish species lacking accurate functional annotation and genomic resources. The workflow allowed to obtain lists of genes supposed to be enriched in true positive differentially expressed genes (DEGs), which were subjected to over-representation analysis methods (ORA). Several pathways and ontologies, mostly related to cell division and metabolism, sexual reproduction and steroid production, were found significantly enriched in our analyses. Moreover, two sets of potential ovotestis biomarkers were selected using several criteria. The first group displayed specific potential biomarkers belonging to pathways/ontologies highlighted in the experiment. Among them, the early ovarian differentiation gene foxl2a was overexpressed. The second group, which was highly sensitive but not specific, included the DEGs presenting the highest fold change and lowest p

  3. Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans.

    PubMed

    Sims, Andrew H; Robson, Geoffrey D; Hoyle, David C; Oliver, Stephen G; Turner, Geoffrey; Prade, Rolf A; Russell, Hugh H; Dunn-Coleman, Nigel S; Gent, Manda E

    2004-02-01

    The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.

  4. Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation.

    PubMed

    Debaugnies, France; Busson, Laurent; Ferster, Alina; Lewalle, Philippe; Azzi, Nadira; Aoun, Mickael; Verhaegen, Godelieve; Mahadeb, Bhavna; de Marchin, Jérôme; Vandenberg, Olivier; Hallin, Marie

    2014-07-01

    Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests = 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test.

  5. Expression profile of long non-coding RNAs in colorectal cancer: A microarray analysis.

    PubMed

    Luo, Jia; Xu, Luning; Jiang, Yigui; Zhuo, Dexiang; Zhang, Shengjun; Wu, Lianhui; Xu, Huadong; Huang, Yue

    2016-04-01

    Colorectal cancer (CRC) is one of the most prevalent malignant tumors and the second cause of cancer-related mortality worldwide. Due to increased morbidity and mortality rates, there is an urgent need to understand the pathogenesis of CRC, discover strategies that can improve diagnosis, and ultimately identify therapies targeting this disease. Over the past several years, research into tumor progression mechanisms has been devoted to identifying and understanding various coding and non-coding regions of the genome and how these genetic variants may affect tumorigenesis and progression. Recently, long non-coding RNAs (lncRNAs), which are non‑protein coding transcripts longer than 200 nucleotides, have emerged as a key aspect in tumor pathogenesis. In the present study, we examined the lncRNA and mRNA expression profiles in 4 patients with colon adenocarcinoma, with paired adjacent normal tissues as controls. Microarray data showed that a total of 3,523 lncRNAs and 2,515 mRNAs were consistently differentially expressed in the CRC tissues compared to adjacent normal tissues. Upon comparison of the differentially expressed transcripts between the groups, we identified 22 pathways which were related to the upregulated transcripts and 24 pathways that corresponded to the downregulated transcripts. Gene ontology analysis revealed that the upregulated transcripts were predominantly enriched in DNA metabolic processes, and the downregulated transcripts were predominantly enriched in organic hydroxyl compound metabolic processes. Coding-non-coding gene co-expression analysis showed that these differentially expressed lncRNAs were closely correlated with 'Wnt signaling pathway' components, whose aberrant activation plays a central role in CRC, indicating that a functional correlation exists between them. In conclusion, the results of the microarray and informatic analysis strongly suggest that lncRNA dysregulation is involved in the complicated process of CRC development

  6. Microarray-Based Analysis of Differential Gene Expression between Infective and Noninfective Larvae of Strongyloides stercoralis

    PubMed Central

    Ramanathan, Roshan; Varma, Sudhir; Ribeiro, José M. C.; Myers, Timothy G.; Nolan, Thomas J.; Abraham, David; Lok, James B.; Nutman, Thomas B.

    2011-01-01

    Background Differences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralis at the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose. Methods and Findings Oligonucleotide hybridization probes for the array were designed to bind 3,571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs) obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 935 differentially expressed genes (469 L3i-biased; 466 L1-biased) having two-fold expression differences or greater and microarray signals with a p value<0.01. Based on a functional analysis, L1 larvae have a larger number of genes putatively involved in transcription (p = 0.004), and L3i larvae have biased expression of putative heat shock proteins (such as hsp-90). Genes with products known to be immunoreactive in S. stercoralis-infected humans (such as SsIR and NIE) had L3i biased expression. Abundantly expressed L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1 and hsp-90, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the 25 most highly expressed L3i genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in L3i biased genes and may be a valuable S. stercoralis target of interest. Conclusions A new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding

  7. 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.

  8. Microarray analysis reveals novel features of the muscle aging process in men and women.

    PubMed

    Liu, Dongmei; Sartor, Maureen A; Nader, Gustavo A; Pistilli, Emidio E; Tanton, Leah; Lilly, Charles; Gutmann, Laurie; IglayReger, Heidi B; Visich, Paul S; Hoffman, Eric P; Gordon, Paul M

    2013-09-01

    To develop a global view of muscle transcriptional differences between older men and women and sex-specific aging, we obtained muscle biopsies from the biceps brachii of young and older men and women and profiled the whole-genome gene expression using microarray. A logistic regression-based method in combination with an intensity-based Bayesian moderated t test was used to identify significant sex- and aging-related gene functional groups. Our analysis revealed extensive sex differences in the muscle transcriptome of older individuals and different patterns of transcriptional changes with aging in men and women. In older women, we observed a coordinated transcriptional upregulation of immune activation, extracellular matrix remodeling, and lipids storage; and a downregulation of mitochondrial biogenesis and function and muscle regeneration. The effect of aging results in sexual dimorphic alterations in the skeletal muscle transcriptome, which may modify the risk for developing musculoskeletal and metabolic diseases in men and women.

  9. Microarray Analysis Reveals Novel Features of the Muscle Aging Process in Men and Women

    PubMed Central

    2013-01-01

    To develop a global view of muscle transcriptional differences between older men and women and sex-specific aging, we obtained muscle biopsies from the biceps brachii of young and older men and women and profiled the whole-genome gene expression using microarray. A logistic regression-based method in combination with an intensity-based Bayesian moderated t test was used to identify significant sex- and aging-related gene functional groups. Our analysis revealed extensive sex differences in the muscle transcriptome of older individuals and different patterns of transcriptional changes with aging in men and women. In older women, we observed a coordinated transcriptional upregulation of immune activation, extracellular matrix remodeling, and lipids storage; and a downregulation of mitochondrial biogenesis and function and muscle regeneration. The effect of aging results in sexual dimorphic alterations in the skeletal muscle transcriptome, which may modify the risk for developing musculoskeletal and metabolic diseases in men and women. PMID:23418191

  10. Component retention in principal component analysis with application to cDNA microarray data

    PubMed Central

    Cangelosi, Richard; Goriely, Alain

    2007-01-01

    Shannon entropy is used to provide an estimate of the number of interpretable components in a principal component analysis. In addition, several ad hoc stopping rules for dimension determination are reviewed and a modification of the broken stick model is presented. The modification incorporates a test for the presence of an "effective degeneracy" among the subspaces spanned by the eigenvectors of the correlation matrix of the data set then allocates the total variance among subspaces. A summary of the performance of the methods applied to both published microarray data sets and to simulated data is given. This article was reviewed by Orly Alter, John Spouge (nominated by Eugene Koonin), David Horn and Roy Varshavsky (both nominated by O. Alter). PMID:17229320

  11. Antimicrobial resistance determinant microarray for analysis of multi-drug resistant isolates

    NASA Astrophysics Data System (ADS)

    Taitt, Chris Rowe; Leski, Tomasz; Stenger, David; Vora, Gary J.; House, Brent; Nicklasson, Matilda; Pimentel, Guillermo; Zurawski, Daniel V.; Kirkup, Benjamin C.; Craft, David; Waterman, Paige E.; Lesho, Emil P.; Bangurae, Umaru; Ansumana, Rashid

    2012-06-01

    The prevalence of multidrug-resistant infections in personnel wounded in Iraq and Afghanistan has made it challenging for physicians to choose effective therapeutics in a timely fashion. To address the challenge of identifying the potential for drug resistance, we have developed the Antimicrobial Resistance Determinant Microarray (ARDM) to provide DNAbased analysis for over 250 resistance genes covering 12 classes of antibiotics. Over 70 drug-resistant bacteria from different geographic regions have been analyzed on ARDM, with significant differences in patterns of resistance identified: genes for resistance to sulfonamides, trimethoprim, chloramphenicol, rifampin, and macrolide-lincosamidesulfonamide drugs were more frequently identified in isolates from sources in Iraq/Afghanistan. Of particular concern was the presence of genes responsible for resistance to many of the last-resort antibiotics used to treat war traumaassociated infections.

  12. RNA Expression Microarray Analysis in Mouse Prospermatogonia: Identification of Candidate Epigenetic Modifiers

    PubMed Central

    Lefèvre, Christophe; Mann, Jeffrey R.

    2011-01-01

    The mammalian totipotent and pluripotent lineage exhibits genome-wide dynamics in respect to DNA methylation content. The first phase of global DNA demethylation and de novo remethylation occurs during preimplantation development and gastrulation, respectively, while the second phase occurs in primordial germ cells and primary oocytes/prospermatogonia, respectively. These dynamics are indicative of a comprehensive epigenetic resetting or reprogramming of the genome in preparation for major differentiation events. To gain further insight into the mechanisms driving DNA methylation dynamics and other types of epigenetic modification, we performed an RNA expression microarray analysis of fetal prospermatogonia at the stage when they are undergoing rapid de novo DNA remethylation. We have identified a number of highly or specifically expressed genes which could be important for determining epigenetic change in prospermatogonia. These data provide a useful resource in the discovery of molecular pathways involved in epigenetic reprogramming in the mammalian germ line. PMID:18330932

  13. Chromosomal Microarray Analysis (CMA) a Clinical Diagnostic Tool in the Prenatal and Postnatal Settings.

    PubMed

    Batzir, Nurit Assia; Shohat, Mordechai; Maya, Idit

    2015-09-01

    Chromosomal microarray analysis (CMA) is a technology used for the detection of clinically-significant microdeietions or duplications, with a high sensitivity for submicroscopic aberrations. It is able to detect changes as small as 5-10Kb in size - a resolution up to 1000 times higher than that of conventional karyotyping. CMA is used for uncovering copy number variants (CNVs) thought to play an important role in the pathogenesis of a variety of disorders, primarily neurodevelopmental disorders and congenital anomalies. CMA may be applied in the prenatal or postnatal setting, with unique benefits and limitations in each setting. The growing use of CMA makes it essential for practicing physicians to understand the principles of this technology and be aware of its powers and limitations.

  14. Parents’ Perceptions of the Usefulness of Chromosomal Microarray Analysis for Children with Autism Spectrum Disorders

    PubMed Central

    Giarelli, Ellen; Bernhardt, Barbara A.; Easley, Ebony; Spinner, Nancy B.; Sankar, Pamela L.; Mulchandani, Surabhi

    2015-01-01

    Clinical guidelines recommend chromosomal microarray analysis (CMA) for all children with autism spectrum disorders (ASDs). We explored the test’s perceived usefulness among parents of children with ASD who had undergone CMA, and received a result categorized as pathogenic, variant of uncertain significance, or negative. Fifty-seven parents participated in a semi-structured telephone interview, and 50 also completed a survey. Most parents reported that CMA was helpful for their child and family. Major themes regarding perceived usefulness were: medical care, educational and behavioral interventions, causal explanation, information for family members, and advancing knowledge. Limits to utility, uncertainties and negative outcomes were also identified. Our findings highlight the importance of considering both health and non-health related utility in genomic testing. PMID:26066358

  15. High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

    PubMed Central

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  16. Current Practice and Utility of Chromosome Microarray Analysis in Infants Undergoing Cardiac Surgery

    PubMed Central

    Buckley, Jason R.; Kavarana, Minoo N.; Chowdhury, Shahryar M.; Scheurer, Mark A.

    2014-01-01

    Objective Traditionally, karyotype and fluorescence in situ hybridization (FISH) were used for cytogenetic testing of infants with congenital heart disease who underwent cardiac surgery at our institution. Recently, chromosome microarray analysis (CMA) has been performed in lieu of the traditional tests. A standardized approach to cytogenetic testing does not exist in this population. The purpose of this study was to assess the utility of CMA based on our current ordering practice. Design We reviewed the records of all infants (< 1 year old) who underwent cardiac surgery at our institution from January 2010 to June 2013. Data included results of all cytogenetic testing performed. Diagnostic yield was calculated as the percentage of significant abnormal results obtained by each test modality. Patients were grouped by classification of congenital heart disease (CHD). Results Two hundred and seventy-five (51%) of 535 infants who underwent cardiac surgery had cytogenetic testing. Of those tested, 154 (56%) had multiple tests performed and at least 18% were redundant or overlapping. The utilization of CMA has increased each year since its implementation. The diagnostic yield for karyotype, FISH and CMA was 10%, 12% and 14% respectively. CMA yield was significantly higher in patients with septal defects (33%, p = 0.01) compared to all other CHD classes. CMA detected abnormalities of unknown clinical significance in 13% of infants tested. Conclusions In our center, redundant cytogenetic testing is frequently performed in infants undergoing cardiac surgery. The utilization of chromosome microarray analysis has increased over time and abnormalities of unknown clinical significance are detected in an important subset of patients. A screening algorithm that risk-stratifies based on classification of CHD and clinical suspicion may provide a practical, data-driven approach to genetic testing in this population and limit unnecessary resource utilization. PMID:25494910

  17. Microarray analysis identifies candidate genes for key roles in coral development

    PubMed Central

    Grasso, Lauretta C; Maindonald, John; Rudd, Stephen; Hayward, David C; Saint, Robert; Miller, David J; Ball, Eldon E

    2008-01-01

    Background Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired. Results Of 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization. Conclusion This study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemone Nematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease. PMID:19014561

  18. Deep sequencing and human antibody repertoire analysis

    PubMed Central

    Boyd, Scott D; Crowe, James E

    2016-01-01

    In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field. PMID:27065089

  19. Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis

    PubMed Central

    Jogia, Gabriella E.; Tronser, Tina; Popova, Anna A.; Levkin, Pavel A.

    2016-01-01

    Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy. PMID:27941668

  20. 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.

  1. Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni

    PubMed Central

    Waisberg, Michael; Lobo, Francisco P; Cerqueira, Gustavo C; Passos, Liana KJ; Carvalho, Omar S; Franco, Glória R; El-Sayed, Najib M

    2007-01-01

    Background The parasitic trematode Schistosoma mansoni is one of the major causative agents of Schistosomiasis, a disease that affects approximately 200 million people, mostly in developing countries. Since much of the pathology is associated with eggs laid by the female worm, understanding the mechanisms involved in oogenesis and sexual maturation is an important step towards the discovery of new targets for effective drug therapy. It is known that the adult female worm only develops fully in the presence of a male worm and that the rates of oviposition and maturation of eggs are significantly increased by mating. In order to study gene transcripts associated with sexual maturation and oviposition, we compared the gene expression profiles of sexually mature and immature parasites using DNA microarrays. Results For each experiment, three amplified RNA microarray hybridizations and their dye swaps were analyzed. Our results show that 265 transcripts are differentially expressed in adult females and 53 in adult males when mature and immature worms are compared. Of the genes differentially expressed, 55% are expressed at higher levels in paired females while the remaining 45% are more expressed in unpaired ones and 56.6% are expressed at higher levels in paired male worms while the remaining 43.4% are more expressed in immature parasites. Real-time RT-PCR analysis validated the microarray results. Several new maturation associated transcripts were identified. Genes that were up-regulated in single-sex females were mostly related to energy generation (i.e. carbohydrate and protein metabolism, generation of precursor metabolites and energy, cellular catabolism, and organelle organization and biogenesis) while genes that were down-regulated related to RNA metabolism, reactive oxygen species metabolism, electron transport, organelle organization and biogenesis and protein biosynthesis. Conclusion Our results confirm previous observations related to gene expression induced

  2. Inverse correlation between CD8+ inflammatory cells and E-cadherin expression in gallbladder cancer: Tissue microarray and imaging analysis

    PubMed Central

    Kai, Keita; Masuda, Masanori; Aishima, Shinichi

    2017-01-01

    AIM To investigated the association between the tumor cells’ expression of E-cadherin and the numbers of several types of inflammatory cells infiltrating into the invasive portion of gallbladder cancer (GBC). METHODS We analyzed 50 GBC cases for which a sufficient amount of tumor tissues for tissue microarray (TMA) had been saved. Three tissue cores (3.0 mm) of invasive lesion from each case were used for the TMA. The 4-μm cut sections on slides were immunostained using primary antibodies including E-cadherin for cancer cells, leukocyte common antigen for leukocyte, myeloperoxidase for neutrophils, CD3 for T cells, CD4 for helper T cells, CD8 for killer T cells, CD20 for B cells and CD68 for macrophages. The immunostained slides were digitally analyzed by imaging analysis software. RESULTS A significant inverse correlation between the number of infiltrating CD8+ cells at invasive areas and the expression of E-cadherin by cancer cells was observed (P = 0.0001), although the degree of this correlation was relatively weak (R = 0.32). The number of CD8+ cells and the cancer cells’ E-cadherin expression were also significantly correlated with tumor differentiation (well-differentiated vs poorly differentiated) (P = 0.0467 and P = 0.0294, respectively). Inverse correlation of T-stage and the number of CD8+ cell infiltration was observed with statistical significance in comparison of T2 and T3 cases (P = 0.0324). CONCLUSION Our findings indicate an inverse correlation of CD8+ T cell infiltration and cancer cells’ E-cadherin expression at invasive areas of GBC. Further analyses are essential to test these findings. PMID:28138440

  3. Transcriptional analysis of the innate immune response using the avian innate immunity microarray

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The avian innate immunity microarray (AIIM) is a genomics tool designed to study the transcriptional activity of the avian immune response (Cytogenet. Genome Res. 117:139-145, 2007). It is an avian cDNA microarray representing 4,959 avian genes spotted in triplicate. The AIIM contains 25 avian int...

  4. Microarray analysis of gene expression in acaricide-exposed Rhipcephalus (Boophilus) microplus larvae.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13,000 probes, having been derived from a previous...

  5. Diagnostic Yield of Chromosomal Microarray Analysis in a Cohort of Patients with Autism Spectrum Disorders from a Highly Consanguineous Population

    ERIC Educational Resources Information Center

    Al-Mamari, Watfa; Al-Saegh, Abeer; Al-Kindy, Adila; Bruwer, Zandre; Al-Murshedi, Fathiya; Al-Thihli, Khalid

    2015-01-01

    Autism Spectrum Disorders are a complicated group of disorders characterized with heterogeneous genetic etiologies. The genetic investigations for this group of disorders have expanded considerably over the past decade. In our study we designed a tired approach and studied the diagnostic yield of chromosomal microarray analysis on patients…

  6. A Meta-Analysis of Microarray Gene Expression in Mouse Stem Cells: Redefining Stemness

    PubMed Central

    Edwards, Yvonne J. K.; Bryson, Kevin; Jones, David T.

    2008-01-01

    Background While much progress has been made in understanding stem cell (SC) function, a complete description of the molecular mechanisms regulating SCs is not yet established. This lack of knowledge is a major barrier holding back the discovery of therapeutic uses of SCs. We investigated the value of a novel meta-analysis of microarray gene expression in mouse SCs to aid the elucidation of regulatory mechanisms common to SCs and particular SC types. Methodology/Principal Findings We added value to previously published microarray gene expression data by characterizing the promoter type likely to regulate transcription. Promoters of up-regulated genes in SCs were characterized in terms of alternative promoter (AP) usage and CpG-richness, with the aim of correlating features known to affect transcriptional control with SC function. We found that SCs have a higher proportion of up-regulated genes using CpG-rich promoters compared with the negative controls. Comparing subsets of SC type with the controls a slightly different story unfolds. The differences between the proliferating adult SCs and the embryonic SCs versus the negative controls are statistically significant. Whilst the difference between the quiescent adult SCs compared with the negative controls is not. On examination of AP usage, no difference was observed between SCs and the controls. However, comparing the subsets of SC type with the controls, the quiescent adult SCs are found to up-regulate a larger proportion of genes that have APs compared to the controls and the converse is true for the proliferating adult SCs and the embryonic SCs. Conclusions/Significance These findings suggest that looking at features associated with control of transcription is a promising future approach for characterizing “stemness” and that further investigations of stemness could benefit from separate considerations of different SC states. For example, “proliferating-stemness” is shown here, in terms of promoter

  7. Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity

    SciTech Connect

    Elferink, M.G.L. Olinga, P.; Draaisma, A.L.; Merema, M.T.; Bauerschmidt, S.; Polman, J.; Schoonen, W.G.; Groothuis, G.M.M.

    2008-06-15

    The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl{sub 4}, fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.

  8. Membrane-based microarrays

    NASA Astrophysics Data System (ADS)

    Dawson, Elliott P.; Hudson, James; Steward, John; Donnell, Philip A.; Chan, Wing W.; Taylor, Richard F.

    1999-11-01

    Microarrays represent a new approach to the rapid detection and identification of analytes. Studies to date have shown that the immobilization of receptor molecules (such as DNA, oligonucleotides, antibodies, enzymes and binding proteins) onto silicon and polymeric substrates can result in arrays able to detect hundreds of analytes in a single step. The formation of the receptor/analyte complex can, itself, lead to detection, or the complex can be interrogated through the use of fluorescent, chemiluminescent or radioactive probes and ligands.

  9. Comparison of three microarray probe annotation pipelines: differences in strategies and their effect on downstream analysis

    PubMed Central

    Neerincx, Pieter BT; Casel, Pierrot; Prickett, Dennis; Nie, Haisheng; Watson, Michael; Leunissen, Jack AM; Groenen, Martien AM; Klopp, Christophe

    2009-01-01

    Background Reliable annotation linking oligonucleotide probes to target genes is essential for functional biological analysis of microarray experiments. We used the IMAD, OligoRAP and sigReannot pipelines to update the annotation for the ARK-Genomics Chicken 20 K array as part of a joined EADGENE/SABRE workshop. In this manuscript we compare their annotation strategies and results. Furthermore, we analyse the effect of differences in updated annotation on functional analysis for an experiment involving Eimeria infected chickens and finally we propose guidelines for optimal annotation strategies. Results IMAD, OligoRAP and sigReannot update both annotation and estimated target specificity. The 3 pipelines can assign oligos to target specificity categories although with varying degrees of resolution. Target specificity is judged based on the amount and type of oligo versus target-gene alignments (hits), which are determined by filter thresholds that users can adjust based on their experimental conditions. Linking oligos to annotation on the other hand is based on rigid rules, which differ between pipelines. For 52.7% of the oligos from a subset selected for in depth comparison all pipelines linked to one or more Ensembl genes with consensus on 44.0%. In 31.0% of the cases none of the pipelines could assign an Ensembl gene to an oligo and for the remaining 16.3% the coverage differed between pipelines. Differences in updated annotation were mainly due to different thresholds for hybridisation potential filtering of oligo versus target-gene alignments and different policies for expanding annotation using indirect links. The differences in updated annotation packages had a significant effect on GO term enrichment analysis with consensus on only 67.2% of the enriched terms. Conclusion In addition to flexible thresholds to determine target specificity, annotation tools should provide metadata describing the relationships between oligos and the annotation assigned to them

  10. TMA Navigator: Network inference, patient stratification and survival analysis with tissue microarray data.

    PubMed

    Lubbock, Alexander L R; Katz, Elad; Harrison, David J; Overton, Ian M

    2013-07-01

    Tissue microarrays (TMAs) allow multiplexed analysis of tissue samples and are frequently used to estimate biomarker protein expression in tumour biopsies. TMA Navigator (www.tmanavigator.org) is an open access web application for analysis of TMA data and related information, accommodating categorical, semi-continuous and continuous expression scores. Non-biological variation, or batch effects, can hinder data analysis and may be mitigated using the ComBat algorithm, which is incorporated with enhancements for automated application to TMA data. Unsupervised grouping of samples (patients) is provided according to Gaussian mixture modelling of marker scores, with cardinality selected by Bayesian information criterion regularization. Kaplan-Meier survival analysis is available, including comparison of groups identified by mixture modelling using the Mantel-Cox log-rank test. TMA Navigator also supports network inference approaches useful for TMA datasets, which often constitute comparatively few markers. Tissue and cell-type specific networks derived from TMA expression data offer insights into the molecular logic underlying pathophenotypes, towards more effective and personalized medicine. Output is interactive, and results may be exported for use with external programs. Private anonymous access is available, and user accounts may be generated for easier data management.

  11. A note on joint versus gene-specific mixed model analysis of microarray gene expression data.

    PubMed

    Hoeschele, Ina; Li, Hua

    2005-04-01

    Currently, linear mixed model analyses of expression microarray experiments are performed either in a gene-specific or global mode. The joint analysis provides more flexibility in terms of how parameters are fitted and estimated and tends to be more powerful than the gene-specific analysis. Here we show how to implement the gene-specific linear mixed model analysis as an exact algorithm for the joint linear mixed model analysis. The gene-specific algorithm is exact, when the mixed model equations can be partitioned into unrelated components: One for all global fixed and random effects and the others for the gene-specific fixed and random effects for each gene separately. This unrelatedness holds under three conditions: (1) any gene must have the same number of replicates or probes on all arrays, but these numbers can differ among genes; (2) the residual variance of the (transformed) expression data must be homogeneous or constant across genes (other variance components need not be homogeneous) and (3) the number of genes in the experiment is large. When these conditions are violated, the gene-specific algorithm is expected to be nearly exact.

  12. Methanotrophic bacteria associated to rice roots: the cultivar effect assessed by T-RFLP and microarray analysis.

    PubMed

    Lüke, Claudia; Bodrossy, Levente; Lupotto, Elisabetta; Frenzel, Peter

    2011-10-01

    Rice plants play a key role in regulating methane emissions from paddy fields by affecting both underlying processes: methane production and oxidation. Specific differences were reported for methane oxidation rates; however, studies on the bacterial communities involved are rare. Here, we analysed the methanotrophic community on the roots of 18 different rice cultivars by pmoA-based terminal restriction fragment length polymorphism (T-RFLP) and microarray analysis. Both techniques showed comparable and consistent results revealing a high diversity dominated by type II and type Ib methanotrophs. pmoA microarrays have been successfully used to study methane-oxidizing bacteria in various environments. However, the microarray's full potential resolving community structure has not been exploited yet. Here, we provide an example on how to include this information into multivariate statistics. The analysis revealed a rice cultivar effect on the methanotroph community composition that could be affiliated to the plant genotype. This effect became only significant by including the specific phylogenetic resolution provided by the microarray into the statistical analysis.

  13. VennMaster: Area-proportional Euler diagrams for functional GO analysis of microarrays

    PubMed Central

    Kestler, Hans A; Müller, André; Kraus, Johann M; Buchholz, Malte; Gress, Thomas M; Liu, Hongfang; Kane, David W; Zeeberg, Barry R; Weinstein, John N

    2008-01-01

    Background Microarray experiments generate vast amounts of data. The functional context of differentially expressed genes can be assessed by querying the Gene Ontology (GO) database via GoMiner. Directed acyclic graph representations, which are used to depict GO categories enriched with differentially expressed genes, are difficult to interpret and, depending on the particular analysis, may not be well suited for formulating new hypotheses. Additional graphical methods are therefore needed to augment the GO graphical representation. Results We present an alternative visualization approach, area-proportional Euler diagrams, showing set relationships with semi-quantitative size information in a single diagram to support biological hypothesis formulation. The cardinalities of sets and intersection sets are represented by area-proportional Euler diagrams and their corresponding graphical (circular or polygonal) intersection areas. Optimally proportional representations are obtained using swarm and evolutionary optimization algorithms. Conclusion VennMaster's area-proportional Euler diagrams effectively structure and visualize the results of a GO analysis by indicating to what extent flagged genes are shared by different categories. In addition to reducing the complexity of the output, the visualizations facilitate generation of novel hypotheses from the analysis of seemingly unrelated categories that share differentially expressed genes. PMID:18230172

  14. Gene expression microarray analysis of early oxygen-induced retinopathy in the rat.

    PubMed

    Tea, Melinda; Fogarty, Rhys; Brereton, Helen M; Michael, Michael Z; Van der Hoek, Mark B; Tsykin, Anna; Coster, Douglas J; Williams, Keryn A

    2009-12-12

    Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

  15. Characterization and analysis of an industrial strain of Streptomyces bingchenggensis by genome sequencing and gene microarray.

    PubMed

    Wang, Xiang-Jing; Zhang, Bo; Yan, Yi-Jun; An, Jing; Zhang, Ji; Liu, Chong-Xi; Xiang, Wen-Sheng

    2013-11-01

    Streptomyces bingchenggensis is a soil bacterium that produces milbemycins, a family of macrolide antibiotics that are commercially important in crop protection and veterinary medicine. In addition, S. bingchenggensis produces many other natural products including the polyether nanchangmycin and novel cyclic pentapeptides. To identify the gene clusters involved in the biosynthesis of these compounds, and better clarify the biochemical pathways of these gene clusters, the whole genome of S. bingchenggensis was sequenced, and the transcriptome profile was subsequently investigated by microarray. In comparison with other sequenced genomes in Streptomyces, S. bingchenggensis has the largest linear chromosome consisting of 11 936 683 base pairs (bp), with an average GC content of 70.8%. The 10 023 predicted protein-coding sequences include at least 47 gene clusters correlated with the biosynthesis of known or predicted secondary metabolites. Transcriptional analysis demonstrated an extremely high expression level of the milbemycin gene cluster during the entire growth period and a moderately high expression level of the nanchangmycin gene cluster during the initial hours that subsequently decreased. However, other gene clusters appear to be silent. The genome-wide analysis of the secondary metabolite gene clusters in S. bingchenggensis, coupled with transcriptional analysis, will facilitate the rational development of high milbemycins-producing strains as well as the discovery of new natural products.

  16. Cancer Classification in Microarray Data using a Hybrid Selective Independent Component Analysis and υ-Support Vector Machine Algorithm

    PubMed Central

    Saberkari, Hamidreza; Shamsi, Mousa; Joroughi, Mahsa; Golabi, Faegheh; Sedaaghi, Mohammad Hossein

    2014-01-01

    Microarray data have an important role in identification and classification of the cancer tissues. Having a few samples of microarrays in cancer researches is always one of the most concerns which lead to some problems in designing the classifiers. For this matter, preprocessing gene selection techniques should be utilized before classification to remove the noninformative genes from the microarray data. An appropriate gene selection method can significantly improve the performance of cancer classification. In this paper, we use selective independent component analysis (SICA) for decreasing the dimension of microarray data. Using this selective algorithm, we can solve the instability problem occurred in the case of employing conventional independent component analysis (ICA) methods. First, the reconstruction error and selective set are analyzed as independent components of each gene, which have a small part in making error in order to reconstruct new sample. Then, some of the modified support vector machine (υ-SVM) algorithm sub-classifiers are trained, simultaneously. Eventually, the best sub-classifier with the highest recognition rate is selected. The proposed algorithm is applied on three cancer datasets (leukemia, breast cancer and lung cancer datasets), and its results are compared with other existing methods. The results illustrate that the proposed algorithm (SICA + υ-SVM) has higher accuracy and validity in order to increase the classification accuracy. Such that, our proposed algorithm exhibits relative improvements of 3.3% in correctness rate over ICA + SVM and SVM algorithms in lung cancer dataset. PMID:25426433

  17. Cancer Classification in Microarray Data using a Hybrid Selective Independent Component Analysis and υ-Support Vector Machine Algorithm.

    PubMed

    Saberkari, Hamidreza; Shamsi, Mousa; Joroughi, Mahsa; Golabi, Faegheh; Sedaaghi, Mohammad Hossein

    2014-10-01

    Microarray data have an important role in identification and classification of the cancer tissues. Having a few samples of microarrays in cancer researches is always one of the most concerns which lead to some problems in designing the classifiers. For this matter, preprocessing gene selection techniques should be utilized before classification to remove the noninformative genes from the microarray data. An appropriate gene selection method can significantly improve the performance of cancer classification. In this paper, we use selective independent component analysis (SICA) for decreasing the dimension of microarray data. Using this selective algorithm, we can solve the instability problem occurred in the case of employing conventional independent component analysis (ICA) methods. First, the reconstruction error and selective set are analyzed as independent components of each gene, which have a small part in making error in order to reconstruct new sample. Then, some of the modified support vector machine (υ-SVM) algorithm sub-classifiers are trained, simultaneously. Eventually, the best sub-classifier with the highest recognition rate is selected. The proposed algorithm is applied on three cancer datasets (leukemia, breast cancer and lung cancer datasets), and its results are compared with other existing methods. The results illustrate that the proposed algorithm (SICA + υ-SVM) has higher accuracy and validity in order to increase the classification accuracy. Such that, our proposed algorithm exhibits relative improvements of 3.3% in correctness rate over ICA + SVM and SVM algorithms in lung cancer dataset.

  18. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

    PubMed

    Pont, M J; Honders, M W; Kremer, A N; van Kooten, C; Out, C; Hiemstra, P S; de Boer, H C; Jager, M J; Schmelzer, E; Vries, R G; Al Hinai, A S; Kroes, W G; Monajemi, R; Goeman, J J; Böhringer, S; Marijt, W A F; Falkenburg, J H F; Griffioen, M

    2016-01-01

    Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

  19. Unique gene expression profile in osteoarthritis synovium compared with cartilage: analysis of publicly accessible microarray datasets.

    PubMed

    Park, Robin; Ji, Jong Dae

    2016-06-01

    The purpose of this study was to identify a gene expression signature in osteoarthritis (OA) synovium and genomic pathways likely to be involved in the pathogenesis of OA. Four publicly accessible microarray studies from synovium of OA patients were integrated, and a transcriptomic and network-based meta-analysis was performed. Based on pathways according to the Kyoto Encyclopedia of Genes and Genomes, functional enrichment analysis was performed. Meta-analysis results of OA synovium were compared to two previously published studies of OA cartilage to determine the relative number of common and specific DEGs of the cartilage and synovium. According to our meta-analysis, a total of 1350 genes were found to be differentially expressed in the synovium of OA patients as compared to that of healthy controls. Pathway analysis found 41 significant pathways in the total DEGs, and 22 and 16 pathways in the upregulated and downregulated DEGs, respectively. Cell adhesion molecules and cytokine-cytokine receptor interaction were the most significant pathway in the upregulated and downregulated DEGs, respectively. Comparison of meta-analysis results of OA synovium with results of two previous studies of OA cartilage identified 85 common genes and 1632 cartilage-specific DEGs and 1265 synovium-specific DEGs in the first study; and 142 common genes, and 856 cartilage-specific DEGs and 1208 synovium-specific DEGs in the second study. Our results show a small overlap between the DEGs of the synovium compared to DEGs of the cartilage, suggesting different pathogenic mechanisms that are specific to the synovium.

  20. Gene set enrichment analysis of microarray data from Pimephales promelas (Rafinesque), a non-mammalian model organism

    PubMed Central

    2011-01-01

    Background Methods for gene-class testing, such as Gene Set Enrichment Analysis (GSEA), incorporate biological knowledge into the analysis and interpretation of microarray data by comparing gene expression patterns to pathways, systems and emergent phenotypes. However, to use GSEA to its full capability with non-mammalian model organisms, a microarray platform must be annotated with human gene symbols. Doing so enables the ability to relate a model organism's gene expression, in response to a given treatment, to potential human health consequences of that treatment. We enhanced the annotation of a microarray platform from a non-mammalian model organism, and then used the GSEA approach in a reanalysis of a study examining the biological significance of acute and chronic methylmercury exposure on liver tissue of fathead minnow (Pimephales promelas). Using GSEA, we tested the hypothesis that fathead livers, in response to methylmercury exposure, would exhibit gene expression patterns similar to diseased human livers. Results We describe an enhanced annotation of the fathead minnow microarray platform with human gene symbols. This resource is now compatible with the GSEA approach for gene-class testing. We confirmed that GSEA, using this enhanced microarray platform, is able to recover results consistent with a previous analysis of fathead minnow exposure to methylmercury using standard analytical approaches. Using GSEA to compare fathead gene expression profiles to human phenotypes, we also found that fathead methylmercury-treated livers exhibited expression profiles that are homologous to human systems & pathways and results in damage that is similar to those of human liver damage associated with hepatocellular carcinoma and hepatitis B. Conclusions This study describes a powerful resource for enabling the use of non-mammalian model organisms in the study of human health significance. Results of microarray gene expression studies involving fathead minnow, typically

  1. Hyperspectral microscopic analysis of normal, benign and carcinoma microarray tissue sections

    NASA Astrophysics Data System (ADS)

    Maggioni, Mauro; Davis, Gustave L.; Warner, Frederick J.; Geshwind, Frank B.; Coppi, Andreas C.; DeVerse, Richard A.; Coifman, Ronald R.

    2006-02-01

    We apply a unique micro-optoelectromechanical tuned light source and new algorithms to the hyper-spectral microscopic analysis of human colon biopsies. The tuned light prototype (Plain Sight Systems Inc.) transmits any combination of light frequencies, range 440nm 700nm, trans-illuminating H and E stained tissue sections of normal (N), benign adenoma (B) and malignant carcinoma (M) colon biopsies, through a Nikon Biophot microscope. Hyper-spectral photomicrographs, randomly collected 400X magnication, are obtained with a CCD camera (Sensovation) from 59 different patient biopsies (20 N, 19 B, 20 M) mounted as a microarray on a single glass slide. The spectra of each pixel are normalized and analyzed to discriminate among tissue features: gland nuclei, gland cytoplasm and lamina propria/lumens. Spectral features permit the automatic extraction of 3298 nuclei with classification as N, B or M. When nuclei are extracted from each of the 59 biopsies the average classification among N, B and M nuclei is 97.1%; classification of the biopsies, based on the average nuclei classification, is 100%. However, when the nuclei are extracted from a subset of biopsies, and the prediction is made on nuclei in the remaining biopsies, there is a marked decrement in performance to 60% across the 3 classes. Similarly the biopsy classification drops to 54%. In spite of these classification differences, which we believe are due to instrument and biopsy normalization issues, hyper-spectral analysis has the potential to achieve diagnostic efficiency needed for objective microscopic diagnosis.

  2. Microarray analysis of human milk cells: persistent high expression of osteopontin during the lactation period

    PubMed Central

    NAGATOMO, T; OHGA, S; TAKADA, H; NOMURA, A; HIKINO, S; IMURA, M; OHSHIMA, K; HARA, T

    2004-01-01

    To continue the search for immunological roles of breast milk, cDNA microarray analysis on cytokines and growth factors was performed for human milk cells. Among the 240 cytokine-related genes, osteopontin (OPN) gene ranked top of the expression. Real-time PCR revealed that the OPN mRNA levels in colostrum cells were approximately 100 times higher than those in PHA-stimulated peripheral blood mononuclear cells (PBMNCs), and 10 000 times higher than those in PB CD14+ cells. The median levels of OPN mRNA in early milk or mature milk cells were more than three times higher than those in colostrum cells. Western blot analysis of human milk showed appreciable expression of full-length and short form proteins of OPN. The concentrations of full-length OPN in early milk or mature milk whey continued to be higher than those in colostrum whey and plasma as assessed by ELISA. The early milk (3–7 days postpartum) contained the highest concentrations of OPN protein, while the late mature milk cells (1 years postpartum) had the highest expression of OPN mRNA of all the lactating periods. The results of immunohistochemical and immunocytochemical staining indicated that OPN-producing epithelial cells and macrophages are found in actively lactating mammary glands. These results suggest that the persistently and extraordinarily high expression of OPN in human milk cells plays a potential role in the immunological development of breast-fed infants. PMID:15373904

  3. Expression Profile of Long Noncoding RNAs in Human Earlobe Keloids: A Microarray Analysis

    PubMed Central

    Guo, Liang; Xu, Kai; Yan, Hongbo; Feng, Haifeng

    2016-01-01

    Background. Long noncoding RNAs (lncRNAs) play key roles in a wide range of biological processes and their deregulation results in human disease, including keloids. Earlobe keloid is a type of pathological skin scar, and the molecular pathogenesis of this disease remains largely unknown. Methods. In this study, microarray analysis was used to determine the expression profiles of lncRNAs and mRNAs between 3 pairs of earlobe keloid and normal specimens. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify the main functions of the differentially expressed genes and earlobe keloid-related pathways. Results. A total of 2068 lncRNAs and 1511 mRNAs were differentially expressed between earlobe keloid and normal tissues. Among them, 1290 lncRNAs and 1092 mRNAs were upregulated, and 778 lncRNAs and 419 mRNAs were downregulated. Pathway analysis revealed that 24 pathways were correlated to the upregulated transcripts, while 11 pathways were associated with the downregulated transcripts. Conclusion. We characterized the expression profiles of lncRNA and mRNA in earlobe keloids and suggest that lncRNAs may serve as diagnostic biomarkers for the therapy of earlobe keloid. PMID:28101509

  4. A Phenotypic microarray analysis of Streptococcus mutans liaS mutant

    PubMed Central

    Zhang, Jiaqin; Biswas, Indranil

    2009-01-01

    Streptococcus mutans, a bioflim-forming gram-positive bacterium that resides in the human oral cavity, is considered to be the primary etiological agent of human dental caries. A cell-envelope stress sensing histidine kinase, LiaS, is considered to be important for expression of virulence factors such as glucan-binding protein C and mutacin production. In this communication, a liaS mutant was subjected to phenotypic microarray (PM) analysis of about 2000 phenotypes that includes utilization of various carbon, nitrogen, phosphate, and sulfur sources; osmolytes; metabolic inhibitors; and susceptibility to toxic compounds, including several types of antibiotics. Compared to the parental strain UA159, the liaS mutant strain (IBS148) was more tolerant to various inhibitors that target protein synthesis, DNA synthesis, and cell-wall biosynthesis. Some of the key findings of the PM analysis were confirmed in independent growth studies and by using antibiotic discs and E-test strips for susceptibility testing. PMID:19118347

  5. A Comprehensive Comparison of Different Clustering Methods for Reliability Analysis of Microarray Data

    PubMed Central

    Kafieh, Rahele; Mehridehnavi, Alireza

    2013-01-01

    In this study, we considered some competitive learning methods including hard competitive learning and soft competitive learning with/without fixed network dimensionality for reliability analysis in microarrays. In order to have a more extensive view, and keeping in mind that competitive learning methods aim at error minimization or entropy maximization (different kinds of function optimization), we decided to investigate the abilities of mixture decomposition schemes. Therefore, we assert that this study covers the algorithms based on function optimization with particular insistence on different competitive learning methods. The destination is finding the most powerful method according to a pre-specified criterion determined with numerical methods and matrix similarity measures. Furthermore, we should provide an indication showing the intrinsic ability of the dataset to form clusters before we apply a clustering algorithm. Therefore, we proposed Hopkins statistic as a method for finding the intrinsic ability of a data to be clustered. The results show the remarkable ability of Rayleigh mixture model in comparison with other methods in reliability analysis task. PMID:24083134

  6. Meta-Analysis of Multiple Sclerosis Microarray Data Reveals Dysregulation in RNA Splicing Regulatory Genes.

    PubMed

    Paraboschi, Elvezia Maria; Cardamone, Giulia; Rimoldi, Valeria; Gemmati, Donato; Spreafico, Marta; Duga, Stefano; Soldà, Giulia; Asselta, Rosanna

    2015-09-30

    Abnormalities in RNA metabolism and alternative splicing (AS) are emerging as important players in complex disease phenotypes. In particular, accumulating evidence suggests the existence of pathogenic links between multiple sclerosis (MS) and altered AS, including functional studies showing that an imbalance in alternatively-spliced isoforms may contribute to disease etiology. Here, we tested whether the altered expression of AS-related genes represents a MS-specific signature. A comprehensive comparative analysis of gene expression profiles of publicly-available microarray datasets (190 MS cases, 182 controls), followed by gene-ontology enrichment analysis, highlighted a significant enrichment for differentially-expressed genes involved in RNA metabolism/AS. In detail, a total of 17 genes were found to be differentially expressed in MS in multiple datasets, with CELF1 being dysregulated in five out of seven studies. We confirmed CELF1 downregulation in MS (p=0.0015) by real-time RT-PCRs on RNA extracted from blood cells of 30 cases and 30 controls. As a proof of concept, we experimentally verified the unbalance in alternatively-spliced isoforms in MS of the NFAT5 gene, a putative CELF1 target. In conclusion, for the first time we provide evidence of a consistent dysregulation of splicing-related genes in MS and we discuss its possible implications in modulating specific AS events in MS susceptibility genes.

  7. Quantitative comparison of the HSV-1 and HSV-2 transcriptomes using DNA microarray analysis

    SciTech Connect

    Aguilar, J.S. . E-mail: jsaguila@uci.edu; Devi-Rao, G.V.; Rice, M.K.; Sunabe, J.; Ghazal, P.; Wagner, E.K.

    2006-04-25

    The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U{sub L}4, U{sub L}29, U{sub L}30, and U{sub L}31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.

  8. Microarray Data Analysis of Space Grown Arabidopsis Leaves for Genes Important in Vascular Patterning

    NASA Technical Reports Server (NTRS)

    Weitzeal, A. J.; Wyatt, S. E.; Parsons-Wingerter, P.

    2016-01-01

    Venation patterning in leaves is a major determinant of photosynthesis efficiency because of its dependency on vascular transport of photoassimilates, water, and minerals. Arabidopsis thaliana grown in microgravity show delayed growth and leaf maturation. Gene expression data from the roots, hypocotyl, and leaves of A. thaliana grown during spaceflight vs. ground control analyzed by Affymetrix microarray are available through NASA's GeneLab (GLDS-7). We analyzed the data for differential expression of genes in leaves resulting from the effects of spaceflight on vascular patterning. Two genes were found by preliminary analysis to be upregulated during spaceflight that may be related to vascular formation. The genes are responsible for coding an ARGOS like protein (potentially affecting cell elongation in the leaves), and an F-box/kelch-repeat protein (possibly contributing to protoxylem specification). Further analysis that will focus on raw data quality assessment and a moderated t-test may further confirm upregulation of the two genes and/or identify other gene candidates. Plants defective in these genes will then be assessed for phenotype by the mapping and quantification of leaf vascular patterning by NASA's VESsel GENeration (VESGEN) software to model specific vascular differences of plants grown in spaceflight.

  9. Microarray Data Analysis of Space Grown Arabidopsis Leaves for Genes Important in Vascular Patterning

    NASA Technical Reports Server (NTRS)

    Weitzeal, A. J.; Wyatt, S. E.; Parsons-Wingerter, P.

    2016-01-01

    Venation patterning in leaves is a major determinant of photosynthesis efficiency because of its dependency on vascular transport of photoassimilates, water, and minerals. Arabidopsis thaliana grown in microgravity show delayed growth and leaf maturation. Gene expression data from the roots, hypocotyl, and leaves of A. thaliana grown during spaceflight vs. ground control analyzed by Affymetrix microarray are available through NASAs GeneLab (GLDS-7). We analyzed the data for differential expression of genes in leaves resulting from the effects of spaceflight on vascular patterning. Two genes were found by preliminary analysis to be upregulated during spaceflight that may be related to vascular formation. The genes are responsible for coding an ARGOS like protein (potentially affecting cell elongation in the leaves), and an F-boxkelch-repeat protein (possibly contributing to protoxylem specification). Further analysis that will focus on raw data quality assessment and a moderated t-test may further confirm upregulation of the two genes and/or identify other gene candidates. Plants defective in these genes will then be assessed for phenotype by the mapping and quantification of leaf vascular patterning by NASAs VESsel GENeration (VESGEN) software to model specific vascular differences of plants grown in spaceflight.

  10. Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

    PubMed Central

    Mercer, Sarah E.; Cheng, Chia-Ho; Atkinson, Donald L.; Krcmery, Jennifer; Guzman, Claudia E.; Kent, David T.; Zukor, Katherine; Marx, Kenneth A.; Odelberg, Shannon J.; Simon, Hans-Georg

    2012-01-01

    The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine. PMID:23300656

  11. Microarray analysis after strenuous exercise in peripheral blood mononuclear cells of endurance horses.

    PubMed

    Capomaccio, S; Cappelli, K; Barrey, E; Felicetti, M; Silvestrelli, M; Verini-Supplizi, A

    2010-12-01

    It is known that moderate physical activity may have beneficial effects on health, whereas strenuous effort induces a state resembling inflammation. The molecular mechanisms underlying the cellular response to exercise remain unclear, although it is clear that the immune system plays a key role. It has been hypothesized that the physio-pathological condition that develops in athletes subjected to heavy training is caused by derangement of cellular immune regulation. The purpose of the present study was to obtain information on endurance horse gene transcription under strenuous conditions and to identify candidate genes causing immune system derangement. We performed a wide gene expression scan, using microarray technology, on peripheral blood mononuclear cells of ten horses chosen from high-level participants in national and international endurance races. The use of three different timepoints revealed changes in gene expression when post-effort samples (T1, taken immediately after the race; and T2, taken 24 h after the race) were compared with basal sample (T0, at rest). Statistical analysis showed no differences in gene expression between T0 and T2 samples, indicating complete restoration of homeostasis by 24 h after racing, whereas T1 showed strong modulation of expression, affecting 132 genes (97 upregulated, 35 downregulated). Ingenuity pathway analysis revealed that the main mechanisms and biofunctions involved were significantly associated with immunological and inflammatory responses. Real-time PCR was performed on 26 gene products to validate the array data.

  12. Microarray analysis of pancreatic gene expression during biotin repletion in biotin-deficient rats.

    PubMed

    Dakshinamurti, Krishnamurti; Bagchi, Rushita A; Abrenica, Bernard; Czubryt, Michael P

    2015-12-01

    Biotin is a B vitamin involved in multiple metabolic pathways. In humans, biotin deficiency is relatively rare but can cause dermatitis, alopecia, and perosis. Low biotin levels occur in individuals with type-2 diabetes, and supplementation with biotin plus chromium may improve blood sugar control. The acute effect on pancreatic gene expression of biotin repletion following chronic deficiency is unclear, therefore we induced biotin deficiency in adult male rats by feeding them a 20% raw egg white diet for 6 weeks. Animals were then randomized into 2 groups: one group received a single biotin supplement and returned to normal chow lacking egg white, while the second group remained on the depletion diet. After 1 week, pancreata were removed from biotin-deficient (BD) and biotin-repleted (BR) animals and RNA was isolated for microarray analysis. Biotin depletion altered gene expression in a manner indicative of inflammation, fibrosis, and defective pancreatic function. Conversely, biotin repletion activated numerous repair and anti-inflammatory pathways, reduced fibrotic gene expression, and induced multiple genes involved in pancreatic endocrine and exocrine function. A subset of the results was confirmed by quantitative real-time PCR analysis, as well as by treatment of pancreatic AR42J cells with biotin. The results indicate that biotin repletion, even after lengthy deficiency, results in the rapid induction of repair processes in the pancreas.

  13. Microarray Analysis of Port Wine Stains Before and After Pulsed Dye Laser Treatment

    PubMed Central

    Laquer, Vivian T.; Hevezi, Peter A.; Albrecht, Huguette; Chen, Tina S.; Zlotnik, Albert; Kelly, Kristen M.

    2014-01-01

    Background and Objectives Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. Materials and Methods Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. Results There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. Conclusion In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were

  14. 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

  15. Analytical Protein Microarrays: Advancements Towards Clinical Applications.

    PubMed

    Sauer, Ursula

    2017-01-29

    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.

  16. A critical comparison of protein microarray fabrication technologies.

    PubMed

    Romanov, Valentin; Davidoff, S Nikki; Miles, Adam R; Grainger, David W; Gale, Bruce K; Brooks, Benjamin D

    2014-03-21

    Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein

  17. How cyanobacteria pose new problems to old methods: challenges in microarray time series analysis

    PubMed Central

    2013-01-01

    Background The transcriptomes of several cyanobacterial strains have been shown to exhibit diurnal oscillation patterns, reflecting the diurnal phototrophic lifestyle of the organisms. The analysis of such genome-wide transcriptional oscillations is often facilitated by the use of clustering algorithms in conjunction with a number of pre-processing steps. Biological interpretation is usually focussed on the time and phase of expression of the resulting groups of genes. However, the use of microarray technology in such studies requires the normalization of pre-processing data, with unclear impact on the qualitative and quantitative features of the derived information on the number of oscillating transcripts and their respective phases. Results A microarray based evaluation of diurnal expression in the cyanobacterium Synechocystis sp. PCC 6803 is presented. As expected, the temporal expression patterns reveal strong oscillations in transcript abundance. We compare the Fourier transformation-based expression phase before and after the application of quantile normalization, median polishing, cyclical LOESS, and least oscillating set (LOS) normalization. Whereas LOS normalization mostly preserves the phases of the raw data, the remaining methods introduce systematic biases. In particular, quantile-normalization is found to introduce a phase-shift of 180°, effectively changing night-expressed genes into day-expressed ones. Comparison of a large number of clustering results of differently normalized data shows that the normalization method determines the result. Subsequent steps, such as the choice of data transformation, similarity measure, and clustering algorithm, only play minor roles. We find that the standardization and the DTF transformation are favorable for the clustering of time series in contrast to the 12 m transformation. We use the cluster-wise functional enrichment of a clustering derived by LOS normalization, clustering using flowClust, and DFT

  18. Microarray analysis of Arabidopsis WRKY33 mutants in response to the necrotrophic fungus Botrytis cinerea

    PubMed Central

    Sham, Arjun; Moustafa, Khaled; Al-Shamisi, Shamma; Alyan, Sofyan; Iratni, Rabah

    2017-01-01

    The WRKY33 transcription factor was reported for resistance to the necrotrophic fungus Botrytis cinerea. Using microarray-based analysis, we compared Arabidopsis WRKY33 overexpressing lines and wrky33 mutant that showed altered susceptibility to B. cinerea with their corresponding wild-type plants. In the wild-type, about 1660 genes (7% of the transcriptome) were induced and 1054 genes (5% of the transcriptome) were repressed at least twofold at early stages of inoculation with B. cinerea, confirming previous data of the contribution of these genes in B. cinerea resistance. In Arabidopsis wild-type plant infected with B. cinerea, the expressions of the differentially expressed genes encoding for proteins and metabolites involved in pathogen defense and non-defense responses, seem to be dependent on a functional WRKY33 gene. The expression profile of 12-oxo-phytodienoic acid- and phytoprostane A1-treated Arabidopsis plants in response to B. cinerea revealed that cyclopentenones can also modulate WRKY33 regulation upon inoculation with B. cinerea. These results support the role of electrophilic oxylipins in mediating plant responses to B. cinerea infection through the TGA transcription factor. Future directions toward the identification of the molecular components in cyclopentenone signaling will elucidate the novel oxylipin signal transduction pathways in plant defense. PMID:28207847

  19. Use of microarray analysis to study gene expression in the avian epiphyseal growth plate.

    PubMed

    Horvat-Gordon, M; Praul, C A; Ramachandran, R; Bartell, P A; Leach, R M

    2010-03-01

    Longitudinal bone growth depends upon the execution of an intricate series of cellular activities by epiphyseal growth plate chondrocytes. In order to better understand these coordinated events, microarray analysis was used to compare gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate. RT-PCR was used to confirm the identity of a select number of genes. The expression of 745 genes was found to differ 3-fold or greater at the 0.05 level of probability. Transferrin was the most highly up-regulated (321-fold) gene associated with chondrocyte hypertrophy. Immunohistochemistry localized this peptide adjacent to the penetrating blood vessels in the growth plate of 3-week-old chicks. Fibulin, OC-116, DMP-1 and PHEX were among the expanded number of genes associated with extracellular matrix metabolism. The presence of NELL2, ATOH8 and PLEXIN suggests a neuronal involvement in growth plate physiology. In addition, the expression of a large number of genes associated with angiogenesis and cellular stress was up-regulated. These processes are important to the physiology and survival of chondrocytes in the unique and stressful environment of the epiphyseal growth plate.

  20. Surface ligation-based resonance light scattering analysis of methylated genomic DNA on a microarray platform.

    PubMed

    Ma, Lan; Lei, Zhen; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-05-10

    DNA methylation is a crucial epigenetic modification and is closely related to tumorigenesis. Herein, a surface ligation-based high throughput method combined with bisulfite treatment is developed for analysis of methylated genomic DNA. In this method, a DNA microarray is employed as a reaction platform, and resonance light scattering (RLS) of nanoparticles is used as the detection principle. The specificity stems from allele-specific ligation of Taq DNA ligase, which is further enhanced by improving the fidelity of Taq DNA ligase in a heterogeneous reaction. Two amplification techniques, rolling circle amplification (RCA) and silver enhancement, are employed after the ligation reaction and a gold nanoparticle (GNP) labeling procedure is used to amplify the signal. As little as 0.01% methylated DNA (i.e. 2 pmol L(-1)) can be distinguished from the cocktail of methylated and unmethylated DNA by the proposed method. More importantly, this method shows good accuracy and sensitivity in profiling the methylation level of genomic DNA of three selected colonic cancer cell lines. This strategy provides a high throughput alternative with reasonable sensitivity and resolution for cancer study and diagnosis.

  1. Identification of Novel Epigenetic Markers of Prostate Cancer by NotI-Microarray Analysis

    PubMed Central

    Dmitriev, Alexey A.; Rosenberg, Eugenia E.; Krasnov, George S.; Gerashchenko, Ganna V.; Gordiyuk, Vasily V.; Pavlova, Tatiana V.; Kudryavtseva, Anna V.; Beniaminov, Artemy D.; Belova, Anastasia A.; Bondarenko, Yuriy N.; Danilets, Rostislav O.; Glukhov, Alexander I.; Kondratov, Aleksandr G.; Alexeyenko, Andrey; Alekseev, Boris Y.; Klein, George; Senchenko, Vera N.; Kashuba, Vladimir I.

    2015-01-01

    A significant need for reliable and accurate cancer diagnostics and prognosis compels the search for novel biomarkers that would be able to discriminate between indolent and aggressive tumors at the early stages of disease. The aim of this work was identification of potential diagnostic biomarkers for characterization of different types of prostate tumors. NotI-microarrays with 180 clones associated with chromosome 3 genes/loci were applied to determine genetic and epigenetic alterations in 33 prostate tumors. For 88 clones, aberrations were detected in more than 10% of tumors. The major types of alterations were DNA methylation and/or deletions. Frequent methylation of the discovered loci was confirmed by bisulfite sequencing on selective sampling of genes: FGF12, GATA2, and LMCD1. Three genes (BHLHE40, BCL6, and ITGA9) were tested for expression level alterations using qPCR, and downregulation associated with hypermethylation was shown in the majority of tumors. Based on these data, we proposed the set of potential biomarkers for detection of prostate cancer and discrimination between prostate tumors with different malignancy and aggressiveness: BHLHE40, FOXP1, LOC285205, ITGA9, CTDSPL, FGF12, LOC440944/SETD5, VHL, CLCN2, OSBPL10/ZNF860, LMCD1, FAM19A4, CAND2, MAP4, KY, and LRRC58. Moreover, we probabilistically estimated putative functional relations between the genes within each set using the network enrichment analysis. PMID:26491211

  2. Microarray analysis of diet-induced alterations in gene expression in the ACI rat prostate.

    PubMed

    Reyes, Niradiz; Iatropoulos, Michael; Mittelman, Abraham; Geliebter, Jan

    2002-08-01

    The natural history of prostate cancer is a multistage process that involves the transition from normal tissue to subclinical cancer, with progression to carcinoma in situ and eventually metastatic disease. Evidence suggests that a high-fat diet plays a critical role in the biology and progression of the disease. ACI rats were maintained for two generations on high beef fat or control diets for 18 months. Affymetrix microarrays were used to analyze the mRNA expression levels in the dorsolateral prostates of rats on the different diets. Approximately 4752 genes and expressed sequence tag (EST) were expressed in the prostates of rats on either diet. Twenty-seven genes were upregulated and 28 genes downregulated in the high beef fat diet. Data analysis indicated that a high beef fat diet affects the expression of genes involved in inflammation, glucose and fatty acid metabolism, androgen metabolism, potential tumor suppression and protein kinase activity, as well as intracellular and extracellular matrix molecules, growth factors and androgen responsive genes. Results from these and future studies will lead to a better understanding of the effect of diet on gene expression in the prostate and facilitate the rational design and assessment of potential dietary programs for prostate cancer prevention.

  3. Microarray analysis in caudal medulla of cattle orally challenged with bovine spongiform encephalopathy.

    PubMed

    Almeida, L M; Basu, U; Williams, J L; Moore, S S; Guan, L L

    2011-10-25

    Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, amphetamine-responsive transcript, and synaptosomal-associated protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.

  4. Knowledge-based analysis of microarray gene expression data by using support vector machines

    SciTech Connect

    William Grundy; Manuel Ares, Jr.; David Haussler

    2001-06-18

    The authors introduce a method of functionally classifying genes by using gene expression data from DNA microarray hybridization experiments. The method is based on the theory of support vector machines (SVMs). SVMs are considered a supervised computer learning method because they exploit prior knowledge of gene function to identify unknown genes of similar function from expression data. SVMs avoid several problems associated with unsupervised clustering methods, such as hierarchical clustering and self-organizing maps. SVMs have many mathematical features that make them attractive for gene expression analysis, including their flexibility in choosing a similarity function, sparseness of solution when dealing with large data sets, the ability to handle large feature spaces, and the ability to identify outliers. They test several SVMs that use different similarity metrics, as well as some other supervised learning methods, and find that the SVMs best identify sets of genes with a common function using expression data. Finally, they use SVMs to predict functional roles for uncharacterized yeast ORFs based on their expression data.

  5. THE SALIVARY TRANSCRIPTOME OF Anopheles gambiae (DIPTERA: CULICIDAE) LARVAE: A MICROARRAY-BASED ANALYSIS

    PubMed Central

    Neira Oviedo, M.; Ribeiro, J.M.C.; Heyland, A.; VanEkeris, L.; Moroz, T.; Linser, P.J.

    2009-01-01

    In spite of the many recent developments in the field of vector sialomics, the salivary glands of larval mosquitoes have been largely unexplored. We used whole-transcriptome microarray analysis to create a gene-expression profile of the salivary gland tissue of fourth-instar Anopheles gambiae larvae, and compare it to the gene-expression profile of a matching group of whole larvae. We identified a total of 221 probes with expression values that were (a) significantly enriched in the salivary glands, and (b) sufficiently annotated as to allow the prediction of the presence/absence of signal peptides in their corresponding gene products. Based on available annotation of the protein sequences associated with these probes, we propose that the main roles of larval salivary secretions include: (a) immune response, (b) mouthpart lubrication, (c) nutrient metabolism, and (d) xenobiotic detoxification. Other highlights of the study include the cloning of a transcript encoding a previously unknown salivary defensin (AgDef5), the confirmation of mucus secretion by the larval salivary glands, and the first report of salivary lipocalins in the Culicidae. PMID:19328852

  6. Protein turnover in atrophying muscle: from nutritional intervention to microarray expression analysis

    NASA Technical Reports Server (NTRS)

    Stein, T. Peter; Wade, Charles E.

    2003-01-01

    PURPOSE OF REVIEW: In response to decreased usage, skeletal muscle undergoes adaptive reductive remodeling due to the decrease in tension on the weight bearing components of the musculo-skeletal system. This response occurs with uncomplicated disuse (e.g. bed rest, space flight), as a secondary consequence of several widely prevalent chronic diseases for which activity is reduced (e.g. chronic obstructive pulmonary disease and chronic heart failure) and is part of the aging process. The problem is therefore one of considerable clinical importance. RECENT FINDINGS: The impaired function and exercise intolerance is related more to the associated muscle wasting rather than to the specific organ system primarily impacted by the disease. Progress has continued in describing the use of anabolic drugs and dietary manipulation. The major advance in the field has been: (i) the discovery of the atrogin-1 gene and (ii) the application of microarray expression analysis and proteomics with the objectives of obtaining comprehensive understanding of the pathways changed with disuse atrophy. SUMMARY: Disuse atrophy is a common clinical problem. There is a need for therapeutic interventions that do not involve exercise. A better understanding of the changes, particularly at the molecular level, could indicate hitherto unsuspected sites for nutritional and pharmacological intervention.

  7. Novel Bifidobacterium promoters selected through microarray analysis lead to constitutive high-level gene expression.

    PubMed

    Wang, Yan; Kim, Jin Yong; Park, Myeong Soo; Ji, Geun Eog

    2012-08-01

    For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.

  8. Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana

    PubMed Central

    Liu, Han-Hua; Tian, Xin; Li, Yan-Jie; Wu, Chang-Ai; Zheng, Cheng-Chao

    2008-01-01

    High-salinity, drought, and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. However, the relationship between miRNA expression and stress responses is just beginning to be explored. Here, we identified 14 stress-inducible miRNAs using microarray data in which the effects of three abiotic stresses were surveyed in Arabidopsis thaliana. Among them, 10 high-salinity-, four drought-, and 10 cold-regulated miRNAs were detected, respectively. miR168, miR171, and miR396 responded to all of the stresses. Expression profiling by RT-PCR analysis showed great cross-talk among the high-salinity, drought, and cold stress signaling pathways. The existence of stress-related elements in miRNA promoter regions provided further evidence supporting our results. These findings extend the current view about miRNA as ubiquitous regulators under stress conditions. PMID:18356539

  9. Genetic counselling and ethical issues with chromosome microarray analysis in prenatal testing.

    PubMed

    McGillivray, George; Rosenfeld, Jill A; McKinlay Gardner, R J; Gillam, Lynn H

    2012-04-01

    Molecular karyotyping using chromosome microarray analysis (CMA) detects more pathogenic chromosomal anomalies than classical karyotyping, making CMA likely to become a first tier test for prenatal diagnosis. Detecting copy number variants of uncertain clinical significance raises ethical considerations. We consider the risk of harm to a woman or her fetus following the detection of a copy number variant of uncertain significance, whether it is ethically justifiable to withhold any test result information from a woman, what constitutes an 'informed choice' when women are offered CMA in pregnancy and whether clinicians are morally responsible for 'unnecessary' termination of pregnancy. Although we are cognisant of the distress associated with uncertain prenatal results, we argue in favour of the autonomy of women and their right to information from genome-wide CMA in order to make informed choices about their pregnancies. We propose that information material to a woman's decision-making process, including uncertain information, should not be withheld, and that it would be paternalistic for clinicians to try to take responsibility for women's decisions to terminate pregnancies. Non-directive pre-test and post-test genetic counselling is central to the delivery of these ethical objectives.

  10. Microarray analysis of New Green Cocoon associated genes in silkworm, Bombyx mori.

    PubMed

    Lu, Ya-Ru; He, Song-Zhen; Tong, Xiao-Ling; Han, Min-Jin; Li, Chun-Lin; Li, Zhi-Quan; Dai, Fang-Yin

    2016-06-01

    Green cocoons in silkworm, Bombyx mori, are caused by flavonoids accumulation in the silk proteins, fibroin and sericin. Despite the economic value of natural green cocoon and medical value of flavonoids, there is limited understanding of the molecular mechanism regulating flavonoids uptake in silkworm, which is tightly associated with the trait of green cocoon. The purpose of this study is to perform a comprehensive analysis to understand the molecular mechanisms of flavonoids uptake in silkworm based on microarray analyses. The study subject was the New Green Cocoon from the silkworm strains, G200 and N100, a new spontaneous dominant green cocoon trait identified in the 2000s. The genes regulating this trait are independent of other green cocoon genes previously reported. Genome-wide gene expression was compared between the New Green Cocoon producing silkworm strains, G200 and N100, and the control sample, which is the white cocoon producing strain 872B. Among these strains, N100 and 872B are near-isogenic lines. The results showed that 130 genes have consistently changing expression patterns in the green cocoon strains when compared with the white cocoon strain. Among these, we focused on the genes related to flavonoids metabolism and absorption, such as sugar transporter genes and UDP-glucosyltransferase genes. Based on our findings, we propose the potential mechanisms for flavonoids absorption and metabolism in silkworm. Our results imply that silkworm might be used as an underlying model for flavonoids in pharmaceutical research.

  11. Microarray Expression Analysis for the Paradoxical Roles of Acanthopanax senticosus Harms in Treating α-Synucleinopathies.

    PubMed

    Li, Xu-zhao; Zhang, Shuai-nan; Lu, Fang; Liu, Shu-min

    2016-02-01

    α-Synuclein is a key player in the pathogenesis of neurodegenerative disorders with Lewy bodies. Our previous studies have also showed that Acanthopanax senticosus harms (AS) could significantly suppress α-synuclein overexpression and toxicity. Identifying the RNAs related to α-synucleinopathies may facilitate understanding the pathogenesis of the diseases and the safe application of AS in the clinic. Microarray expression profiling of long non-coding RNAs (lncRNAs) and mRNAs was undertaken in control non-transgenic and human α-synuclein transgenic mice. The effects of AS on central nervous system (CNS) in pathology and physiology were investigated based on the lncRNA/mRNA targets analysis. In total, 341 lncRNAs and 279 mRNAs were differentially expressed by α-synuclein stimulus, among which 29 lncRNAs and 25 mRNAs were involved in the anti-α-synucleinopathies mechanism of AS. However, the levels of 19/29 lncRNAs and 12/25 mRNAs in AS group were similar to those in α-synuclein group, which may cause potential neurotoxicity analogous to α-synuclein. This study demonstrated that some of lncRNAs/mRNAs were involved in α-synuclein related pathophysiology, and AS produced the bidirectional effects on CNS under pathological and physiological conditions.

  12. Predicting MicroRNA Biomarkers for Cancer Using Phylogenetic Tree and Microarray Analysis

    PubMed Central

    Wang, Hsiuying

    2016-01-01

    MicroRNAs (miRNAs) are shown to be involved in the initiation and progression of cancers in the literature, and the expression of miRNAs is used as an important cancer prognostic tool. The aim of this study is to predict high-confidence miRNA biomarkers for cancer. We adopt a method that combines miRNA phylogenetic structure and miRNA microarray data analysis to discover high-confidence miRNA biomarkers for colon, prostate, pancreatic, lung, breast, bladder and kidney cancers. There are 53 miRNAs selected through this method that either have potential to involve a single cancer’s development or to involve several cancers’ development. These miRNAs can be used as high-confidence miRNA biomarkers of these seven investigated cancers for further experiment validation. miR-17, miR-20, miR-106a, miR-106b, miR-92, miR-25, miR-16, miR-195 and miR-143 are selected to involve a single cancer’s development in these seven cancers. They have the potential to be useful miRNA biomarkers when the result can be confirmed by experiments. PMID:27213352

  13. Characterization and microarray analysis of genes in human lymphatic endothelial cells from patients with breast cancer.

    PubMed

    Kawai, Yoshiko; Minami, Takashi; Fujimori, Minoru; Hosaka, Kayoko; Mizuno, Risuke; Ikomi, Fumitaka; Kodama, Tatsuhiko; Ohhashi, Toshio

    2007-01-01

    We successfully isolated human lymphatic endothelial cells from afferent lymph vessels (HALEC) of sentinel lymph nodes in patients with breast cancer by using trypsin digestion. The cells were cultured in EGM-2 medium with 10% FBS under the condition of 5% O2, 5% CO2, and 90% N2 at 37 degrees C. The cultured cells exhibited a monolayer with cobblestone appearance and a marked phagocytosis of Dil-Ac-LDL. Immunohistochemical lymphatic vessel markers were also found, such as podoplanin, LYVE-1, VEGF receptor 3, and Prox-1. Quantitative RT-PCR analysis also showed that podoplanin, VEGF R3, and Prox-1 mRNA were expressed more selectively in the cultured cells. The cells had marked immunoreactivity to antisera of ecNOS, iNOS, COX1, and weak reactivity of COX2. Constitutively expressed cell-type specific genes of the cultured cells were also analyzed by oligonucleotide microarray methods. Compared with human umbilical vein endothelial cells (HUVEC), the cells selectively expressed 88 known genes such as angiopoietin-like 4, oxygen radicals-related enzymes, and adhesion molecules and the related proteoglycans. The findings suggest that the cultured cells seem to be human lymphatic endothelial cells. In conclusion, the isolated, cannulated and enzymatic digested method we adopted for culture of human lymphatic endothelial cells may be easy and useful for investigating cellular, molecular biological, and genomic properties of the cells.

  14. Meta-Analysis of Microarray Data of Rainbow Trout Fry Gonad Differentiation Modulated by Ethynylestradiol

    PubMed Central

    Depiereux, Sophie; Le Gac, Florence; De Meulder, Bertrand; Pierre, Michael; Helaers, Raphaël; Guiguen, Yann; Kestemont, Patrick; Depiereux, Eric

    2015-01-01

    Sex differentiation in fish is a highly labile process easily reversed by the use of exogenous hormonal treatment and has led to environmental concerns since low doses of estrogenic molecules can adversely impact fish reproduction. The goal of this study was to identify pathways altered by treatment with ethynylestradiol (EE2) in developing fish and to find new target genes to be tested further for their possible role in male-to-female sex transdifferentiation. To this end, we have successfully adapted a previously developed bioinformatics workflow to a meta-analysis of two datasets studying sex reversal following exposure to EE2 in juvenile rainbow trout. The meta-analysis consisted of retrieving the intersection of the top gene lists generated for both datasets, performed at different levels of stringency. The intersecting gene lists, enriched in true positive differentially expressed genes (DEGs), were subjected to over-representation analysis (ORA) which allowed identifying several statistically significant enriched pathways altered by EE2 treatment and several new candidate pathways, such as progesterone-mediated oocyte maturation and PPAR signalling. Moreover, several relevant key genes potentially implicated in the early transdifferentiation process were selected. Altogether, the results show that EE2 has a great effect on gene expression in juvenile rainbow trout. The feminization process seems to result from the altered transcription of genes implicated in normal female gonad differentiation, resulting in expression similar to that observed in normal females (i.e. the repression of key testicular markers cyp17a1, cyp11b, tbx1), as well as from other genes (including transcription factors) that respond specifically to the EE2 treatment. The results also showed that the bioinformatics workflow can be applied to different types of microarray platforms and could be generalized to (eco)toxicogenomics studies for environmental risk assessment purposes. PMID

  15. Identification of tumor epithelium and stroma in tissue microarrays using texture analysis

    PubMed Central

    2012-01-01

    Background The aim of the study was to assess whether texture analysis is feasible for automated identification of epithelium and stroma in digitized tumor tissue microarrays (TMAs). Texture analysis based on local binary patterns (LBP) has previously been used successfully in applications such as face recognition and industrial machine vision. TMAs with tissue samples from 643 patients with colorectal cancer were digitized using a whole slide scanner and areas representing epithelium and stroma were annotated in the images. Well-defined images of epithelium (n = 41) and stroma (n = 39) were used for training a support vector machine (SVM) classifier with LBP texture features and a contrast measure C (LBP/C) as input. We optimized the classifier on a validation set (n = 576) and then assessed its performance on an independent test set of images (n = 720). Finally, the performance of the LBP/C classifier was evaluated against classifiers based on Haralick texture features and Gabor filtered images. Results The proposed approach using LPB/C texture features was able to correctly differentiate epithelium from stroma according to texture: the agreement between the classifier and the human observer was 97 per cent (kappa value = 0.934, P < 0.0001) and the accuracy (area under the ROC curve) of the LBP/C classifier was 0.995 (CI95% 0.991-0.998). The accuracy of the corresponding classifiers based on Haralick features and Gabor-filter images were 0.976 and 0.981 respectively. Conclusions The method illustrates the capability of automated segmentation of epithelial and stromal tissue in TMAs based on texture features and an SVM classifier. Applications include tissue specific assessment of gene and protein expression, as well as computerized analysis of the tumor microenvironment. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4123422336534537 PMID:22385523

  16. Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis.

    PubMed

    Rioja, Inmaculada; Clayton, Chris L; Graham, Simon J; Life, Paul F; Dickson, Marion C

    2005-01-01

    Experimental arthritis models are considered valuable tools for delineating mechanisms of inflammation and autoimmune phenomena. Use of microarray-based methods represents a new and challenging approach that allows molecular dissection of complex autoimmune diseases such as arthritis. In order to characterize the temporal gene expression profile in joints from the reactivation model of streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats, total RNA was extracted from ankle joints from naive, SCW injected, or phosphate buffered saline injected animals (time course study) and gene expression was analyzed using Affymetrix oligonucleotide microarray technology (RAE230A). After normalization and statistical analysis of data, 631 differentially expressed genes were sorted into clusters based on their levels and kinetics of expression using Spotfire profile search and K-mean cluster analysis. Microarray-based data for a subset of genes were validated using real-time PCR TaqMan analysis. Analysis of the microarray data identified 631 genes (441 upregulated and 190 downregulated) that were differentially expressed (Delta > 1.8, P < 0.01), showing specific levels and patterns of gene expression. The genes exhibiting the highest fold increase in expression on days -13.8, -13, or 3 were involved in chemotaxis, inflammatory response, cell adhesion and extracellular matrix remodelling. Transcriptome analysis identified 10 upregulated genes (Delta > 5), which have not previously been associated with arthritis pathology and are located in genomic regions associated with autoimmune disease. The majority of the downregulated genes were associated with metabolism, transport and regulation of muscle development. In conclusion, the present study describes the temporal expression of multiple disease-associated genes with potential pathophysiological roles in the reactivation model of SCW-induced arthritis in Lewis (LEW/N) rat. These findings improve our understanding of

  17. Microarray Analysis of Differential Gene Expression Profile in Peripheral Blood Cells of Patients with Human Essential Hypertension

    PubMed Central

    Korkor, Melvin T.; Meng, Fan Bo; Xing, Shen Yang; Zhang, Mu Chun; Guo, Jin Rui; Zhu, Xiao Xue; Yang, Ping

    2011-01-01

    The polygenic nature of essential hypertension and its dependence on environmental factors pose a challenge for biomedical research. We hypothesized that the analysis of gene expression profiles from peripheral blood cells would distinguish patients with hypertension from normotensives. In order to test this, total RNA from peripheral blood cells was isolated. RNA was reversed-transcribed and labeled and gene expression analyzed using significance Analysis Microarrays (Stanford University, CA, USA). Briefly, Significance Analysis Microarrays (SAM) thresholding identified 31 up-regulated and 18 down-regulated genes with fold changes of ≥2 or≤0.5 and q-value ≤5 % in expression. Statistically significantly gene ontology (GO) function and biological process differentially expressed in essential hypertension were MHC class II receptor activity and immune response respectively. Biological pathway analysis identified several related pathways which are associated with immune/inflammatory responses. Quantitative Real- Time RT-PCR results were consistent with the microarray results. The levels of C - reactive protein were higher in hypertensive patients than normotensives and inflammation-related genes were increased as well. In conclusion, genes enriched for “immune/inflammatory responses” may be associated with essential hypertension. In addition, there is a correlation between systemic inflammation and hypertension. It is anticipated that these findings may provide accurate and efficient strategies for prevention, diagnosis and control of this disorder. PMID:21369372

  18. Co-expression network analysis of Down's syndrome based on microarray data

    PubMed Central

    Zhao, Jianping; Zhang, Zhengguo; Ren, Shumin; Zong, Yanan; Kong, Xiangdong

    2016-01-01

    Down's syndrome (DS) is a type of chromosome disease. The present study aimed to explore the underlying molecular mechanisms of DS. GSE5390 microarray data downloaded from the gene expression omnibus database was used to identify differentially expressed genes (DEGs) in DS. Pathway enrichment analysis of the DEGs was performed, followed by co-expression network construction. Significant differential modules were mined by mutual information, followed by functional analysis. The accuracy of sample classification for the significant differential modules of DEGs was evaluated by leave-one-out cross-validation. A total of 997 DEGs, including 638 upregulated and 359 downregulated genes, were identified. Upregulated DEGs were enriched in 15 pathways, such as cell adhesion molecules, whereas downregulated DEGs were enriched in maturity onset diabetes of the young. Three significant differential modules with the highest discriminative scores (mutual information>0.35) were selected from a co-expression network. The classification accuracy of GSE16677 expression profile samples was 54.55% and 72.73% when characterized by 12 DEGs and 3 significant differential modules, respectively. Genes in significant differential modules were significantly enriched in 5 functions, including the endoplasmic reticulum (P=0.018) and regulation of apoptosis (P=0.061). The identified DEGs, in particular the 12 DEGs in the significant differential modules, such as B-cell lymphoma 2-associated transcription factor 1, heat shock protein 90 kDa beta member 1, UBX domain-containing protein 2 and transmembrane protein 50B, may serve important roles in the pathogenesis of DS. PMID:27588071

  19. Clinical Implementation of Chromosomal Microarray Analysis: Summary of 2513 Postnatal Cases

    PubMed Central

    Lu, Xinyan; Shaw, Chad A.; Patel, Ankita; Li, Jiangzhen; Cooper, M. Lance; Wells, William R.; Sullivan, Cathy M.; Sahoo, Trilochan; Yatsenko, Svetlana A.; Bacino, Carlos A.; Stankiewicz, Pawel; Ou, Zhishu; Chinault, A. Craig; Beaudet, Arthur L.; Lupski, James R.; Cheung, Sau W.; Ward, Patricia A.

    2007-01-01

    Background Array Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detect genomic imbalances and study disease mechanism and pathogenesis. We report our experience with the clinical implementation of this high resolution human genome analysis, referred to as Chromosomal Microarray Analysis (CMA). Methods and Findings CMA was performed clinically on 2513 postnatal samples from patients referred with a variety of clinical phenotypes. The initial 775 samples were studied using CMA array version 4 and the remaining 1738 samples were analyzed with CMA version 5 containing expanded genomic coverage. Overall, CMA identified clinically relevant genomic imbalances in 8.5% of patients: 7.6% using V4 and 8.9% using V5. Among 117 cases referred for additional investigation of a known cytogenetically detectable rearrangement, CMA identified the majority (92.5%) of the genomic imbalances. Importantly, abnormal CMA findings were observed in 5.2% of patients (98/1872) with normal karyotypes/FISH results, and V5, with expanded genomic coverage, enabled a higher detection rate in this category than V4. For cases without cytogenetic results available, 8.0% (42/524) abnormal CMA results were detected; again, V5 demonstrated an increased ability to detect abnormality. Improved diagnostic potential of CMA is illustrated by 90 cases identified with 51 cryptic microdeletions and 39 predicted apparent reciprocal microduplications in 13 specific chromosomal regions associated with 11 known genomic disorders. In addition, CMA identified copy number variations (CNVs) of uncertain significance in 262 probands; however, parental studies usually facilitated clinical interpretation. Of these, 217 were interpreted as familial variants and 11 were determined to be de novo; the remaining 34 await parental studies to resolve the clinical significance. Conclusions This large set of clinical results demonstrates the significantly improved sensitivity of CMA for the

  20. Multievidence microarray mining.

    PubMed

    Seifert, Martin; Scherf, Matthias; Epple, Anton; Werner, Thomas

    2005-10-01

    Microarray mining is a challenging task because of the superposition of several processes in the data. We believe that the combination of microarray data-based analyses (statistical significance analysis of gene expression) with array-independent analyses (literature-mining and promoter analysis) enables some of the problems of traditional array analysis to be overcome. As a proof-of-principle, we revisited publicly available microarray data derived from an experiment with platelet-derived growth factor (PDGF)-stimulated fibroblasts. Our strategy revealed results beyond the detection of the major metabolic pathway known to be linked to the PDGF response: we were able to identify the crosstalking regulatory networks underlying the metabolic pathway without using a priori knowledge about the experiment.

  1. A Grid-based solution for management and analysis of microarrays in distributed experiments

    PubMed Central

    Porro, Ivan; Torterolo, Livia; Corradi, Luca; Fato, Marco; Papadimitropoulos, Adam; Scaglione, Silvia; Schenone, Andrea; Viti, Federica

    2007-01-01

    Several systems have been presented in the last years in order to manage the complexity of large microarray experiments. Although good results have been achieved, most systems tend to lack in one or more fields. A Grid based approach may provide a shared, standardized and reliable solution for storage and analysis of biological data, in order to maximize the results of experimental efforts. A Grid framework has been therefore adopted due to the necessity of remotely accessing large amounts of distributed data as well as to scale computational performances for terabyte datasets. Two different biological studies have been planned in order to highlight the benefits that can emerge from our Grid based platform. The described environment relies on storage services and computational services provided by the gLite Grid middleware. The Grid environment is also able to exploit the added value of metadata in order to let users better classify and search experiments. A state-of-art Grid portal has been implemented in order to hide the complexity of framework from end users and to make them able to easily access available services and data. The functional architecture of the portal is described. As a first test of the system performances, a gene expression analysis has been performed on a dataset of Affymetrix GeneChip® Rat Expression Array RAE230A, from the ArrayExpress database. The sequence of analysis includes three steps: (i) group opening and image set uploading, (ii) normalization, and (iii) model based gene expression (based on PM/MM difference model). Two different Linux versions (sequential and parallel) of the dChip software have been developed to implement the analysis and have been tested on a cluster. From results, it emerges that the parallelization of the analysis process and the execution of parallel jobs on distributed computational resources actually improve the performances. Moreover, the Grid environment have been tested both against the possibility of

  2. Microarray R-based analysis of complex lysate experiments with MIRACLE

    PubMed Central

    List, Markus; Block, Ines; Pedersen, Marlene Lemvig; Christiansen, Helle; Schmidt, Steffen; Thomassen, Mads; Tan, Qihua; Baumbach, Jan; Mollenhauer, Jan

    2014-01-01

    Motivation: Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. Typical challenges involved in this technology are antibody selection, sample preparation and optimization of staining conditions. The issue of combining effective sample management and data analysis, however, has been widely neglected. Results: This motivated us to develop MIRACLE, a comprehensive and user-friendly web application bridging the gap between spotting and array analysis by conveniently keeping track of sample information. Data processing includes correction of staining bias, estimation of protein concentration from response curves, normalization for total protein amount per sample and statistical evaluation. Established analysis methods have been integrated with MIRACLE, offering experimental scientists an end-to-end solution for sample management and for carrying out data analysis. In addition, experienced users have the possibility to export data to R for more complex analyses. MIRACLE thus has the potential to further spread utilization of RPPAs as an emerging technology for high-throughput protein analysis. Availability: Project URL: http://www.nanocan.org/miracle/ Contact: mlist@health.sdu.dk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161257

  3. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  4. A MICROARRAY ANALYSIS OF GENE EXPRESSION IN THE EMBRYONIC FORELIMB OF THE C57BL/6J MOUSE REVEALS SIGNIFICANT ALTERATIONS METABOLIC AND DEVELOPMENTAL REGULATION FOLLOWING ETHANOL EXPOSURE.

    EPA Science Inventory

    The observation of transcriptional changes following embryonic ethanol exposure may provide significant insights into the biological response to ethanol exposure. In this study, we used microarray analysis to examine the transcriptional response of the developing limb to a dose ...

  5. MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

    PubMed

    Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

    2017-01-18

    Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction- Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments.

  6. Transcriptomic Analysis of Trout Gill Ionocytes in Fresh Water and Sea Water Using Laser Capture Microdissection Combined with Microarray Analysis.

    PubMed

    Leguen, Isabelle; Le Cam, Aurélie; Montfort, Jérôme; Peron, Sandrine; Fautrel, Alain

    2015-01-01

    Fish gills represent a complex organ composed of several cell types that perform multiple physiological functions. Among these cells, ionocytes are implicated in the maintenance of ion homeostasis. However, because the ionocyte represents only a small percent of whole gill tissue, its specific transcriptome can be overlooked among the numerous cell types included in the gill. The objective of this study is to better understand ionocyte functions by comparing the RNA expression of this cell type in freshwater and seawater acclimated rainbow trout. To realize this objective, ionocytes were captured from gill cryosections using laser capture microdissection after immunohistochemistry. Then, transcriptome analyses were performed on an Agilent trout oligonucleotide microarray. Gene expression analysis identified 108 unique annotated genes differentially expressed between freshwater and seawater ionocytes, with a fold change higher than 3. Most of these genes were up-regulated in freshwater cells. Interestingly, several genes implicated in ion transport, extracellular matrix and structural cellular proteins appeared up-regulated in freshwater ionocytes. Among them, several ion transporters, such as CIC2, SLC26A6, and NBC, were validated by qPCR and/or in situ hybridization. The latter technique allowed us to localize the transcripts of these ion transporters in only ionocytes and more particularly in the freshwater cells. Genes involved in metabolism and also several genes implicated in transcriptional regulation, cell signaling and the cell cycle were also enhanced in freshwater ionocytes. In conclusion, laser capture microdissection combined with microarray analysis allowed for the determination of the transcriptional signature of scarce cells in fish gills, such as ionocytes, and aided characterization of the transcriptome of these cells in freshwater and seawater acclimated trout.

  7. Microarray Analysis of Gene Expression in Soybean Roots Susceptible to the Soybean Cyst Nematode Two Days Post Invasion

    PubMed Central

    Khan, R.; Alkharouf, N.; Beard, H.; MacDonald, M.; Chouikha, I.; Meyer, S.; Grefenstette, J.; Knap, H.; Matthews, B.

    2004-01-01

    Soybean root cells undergo dramatic morphological and biochemical changes during the establishment of a feeding site in a compatible interaction with the soybean cyst nematode (SCN). We constructed a cDNA microarray with approximately 1,300 cDNA inserts targeted to identify differentially expressed genes during the compatible interaction of SCN with soybean roots 2 days after infection. Three independent biological replicates were grown and inoculated with SCN, and 2 days later RNA was extracted for hybridization to microarrays and compared to noninoculated controls. Statistical analysis indicated that approximately 8% of the genes monitored were induced and more than 50% of these were genes of unknown function. Notable genes that were more highly expressed 2 days after inoculation with SCN as compared to noninoculated roots included the repetitive proline-rich glycoprotein, the stress-induced gene SAM22, ß-1,3-endoglucanase, peroxidase, and those involved in carbohydrate metabolism, plant defense, and signaling. PMID:19262812

  8. Establishment of a reborn MMV-microarray technology: realization of microbiome analysis and other hitherto inaccessible technologies

    PubMed Central

    2014-01-01

    Background With the accelerating development of bioscience, the problem of research cost has become important. We previously devised and developed a novel concept microarray with manageable volumes (MMV) using a soft gel. It demonstrated the great potential of the MMV technology with the examples of 1024-parallel-cell culture and PCR experiments. However, its full potential failed to be expressed, owing to the nature of the material used for the MMV chip. Results In the present study, by developing plastic-based MMVs and associated technologies, we introduced novel technologies such as C2D2P (in which the cells in each well are converted from DNA to protein in 1024-parallel), NGS-non-dependent microbiome analysis, and other powerful applications. Conclusions The reborn MMV-microarray technology has proven to be highly efficient and cost-effective (with approximately 100-fold cost reduction) and enables us to realize hitherto unattainable technologies. PMID:25141858

  9. Aligning ontologies and integrating textual evidence for pathway analysis of microarray data

    SciTech Connect

    Gopalan, Banu; Posse, Christian; Sanfilippo, Antonio P.; Stenzel-Poore, Mary; Stevens, S.L.; Castano, Jose; Beagley, Nathaniel; Riensche, Roderick M.; Baddeley, Bob; Simon, R.P.; Pustejovsky, James

    2006-10-08

    Expression arrays are introducing a paradigmatic change in biology by shifting experimental approaches from single gene studies to genome-level analysis, monitoring the ex-pression levels of several thousands of genes in parallel. The massive amounts of data obtained from the microarray data needs to be integrated and interpreted to infer biological meaning within the context of information-rich pathways. In this paper, we present a methodology that integrates textual information with annotations from cross-referenced ontolo-gies to map genes to pathways in a semi-automated way. We illustrate this approach and compare it favorably to other tools by analyzing the gene expression changes underlying the biological phenomena related to stroke. Stroke is the third leading cause of death and a major disabler in the United States. Through years of study, researchers have amassed a significant knowledge base about stroke, and this knowledge, coupled with new technologies, is providing a wealth of new scientific opportunities. The potential for neu-roprotective stroke therapy is enormous. However, the roles of neurogenesis, angiogenesis, and other proliferative re-sponses in the recovery process following ischemia and the molecular mechanisms that lead to these processes still need to be uncovered. Improved annotation of genomic and pro-teomic data, including annotation of pathways in which genes and proteins are involved, is required to facilitate their interpretation and clinical application. While our approach is not aimed at replacing existing curated pathway databases, it reveals multiple hidden relationships that are not evident with the way these databases analyze functional groupings of genes from the Gene Ontology.

  10. Chromosomal microarray analysis as a first-tier clinical diagnostic test: Estonian experience.

    PubMed

    Zilina, Olga; Teek, Rita; Tammur, Pille; Kuuse, Kati; Yakoreva, Maria; Vaidla, Eve; Mölter-Väär, Triin; Reimand, Tiia; Kurg, Ants; Ounap, Katrin

    2014-03-01

    Chromosomal microarray analysis (CMA) is now established as the first-tier cytogenetic diagnostic test for fast and accurate detection of chromosomal abnormalities in patients with developmental delay/intellectual disability (DD/ID), multiple congenital anomalies (MCA), and autism spectrum disorders (ASD). We present our experience with using CMA for postnatal and prenatal diagnosis in Estonian patients during 2009-2012. Since 2011, CMA is on the official service list of the Estonian Health Insurance Fund and is performed as the first-tier cytogenetic test for patients with DD/ID, MCA or ASD. A total of 1191 patients were analyzed, including postnatal (1072 [90%] patients and 59 [5%] family members) and prenatal referrals (60 [5%] fetuses). Abnormal results were reported in 298 (25%) patients, with a total of 351 findings (1-3 per individual): 147 (42%) deletions, 106 (30%) duplications, 89 (25%) long contiguous stretches of homozygosity (LCSH) events (>5 Mb), and nine (3%) aneuploidies. Of all findings, 143 (41%) were defined as pathogenic or likely pathogenic; for another 143 findings (41%), most of which were LCSH, the clinical significance remained unknown, while 61 (18%) reported findings can now be reclassified as benign or likely benign. Clinically relevant findings were detected in 126 (11%) patients. However, the proportion of variants of unknown clinical significance was quite high (41% of all findings). It seems that our ability to detect chromosomal abnormalities has far outpaced our ability to understand their role in disease. Thus, the interpretation of CMA findings remains a rather difficult task requiring a close collaboration between clinicians and cytogeneticists.

  11. Prenatal sonographic diagnosis of urorectal septum malformation sequence and chromosomal microarray analysis

    PubMed Central

    Pei, Yan; Wu, Qingqing; Liu, Yan; Sun, Lijuan; Zhi, Wenxue; Zhang, Puqing

    2016-01-01

    Abstract Introduction: Urorectal septum malformation sequence (URSMS) is a rare congenital abnormal syndrome that is caused by the incomplete division of the cloaca. Based on whether the cloaca membrane breaks down or not, the URSMS are classified as full and partial forms. The prenatal diagnosis of URSMS remains challenging because of poor recognition to this malformation and the relatively non-specific sonographic features. We report a prenatally sonographic diagnosed case of the partial URSMS, and review the literature to summarize the prenatal features. Case report and review: A 37-year old woman was referred at 24 weeks of gestation for fetal abdominal cyst. Detailed sonographic examination was done and revealed the vesicocolic fistula, distended colon, absence of perianal hypoechoic ring, pyelectasis, and small stomach bubble. The URSMS was suspected. Amniocentesis was done and karyotyping revealed 46,XY. Furthermore, chromosomal microarray analysis (CMA) was performed for the first time in URSMS and an alteration of 111.8Kb deletion was detected in 16p13.3 which was located inside the RBFOX1 gene. Parental studies showed that the deletion was inherited from the father who has nomal clinical phenotype. The woman elected to terminate the pregnancy at 25 weeks gestation and postmortem examination confirmed the diagnosis of partial URSMS. The published studies were reviewed and 28 cases of URSMS with conducted prenatal ultrasonography were collected in this report. The most common sonographic description, as suspicious signs of URSMS, were severe oligohydramnios or anhydramnios, urinary tract anomalies, fetal intra-abdominal cysts, and dilated bowel. Also, enterolithiasis and vesicocolic fistula were relatively infrequent but highly specific feature of URSMS. Conclusions: URSMS is difficult to be diagnosed prenatally. However, it has characteristic features that can be detected by fetal ultrasonography, and a precise prenatal sonographic examination is crucial

  12. Chromosomal microarray analysis as a first-tier clinical diagnostic test: Estonian experience

    PubMed Central

    Žilina, Olga; Teek, Rita; Tammur, Pille; Kuuse, Kati; Yakoreva, Maria; Vaidla, Eve; Mölter-Väär, Triin; Reimand, Tiia; Kurg, Ants; Õunap, Katrin

    2014-01-01

    Chromosomal microarray analysis (CMA) is now established as the first-tier cytogenetic diagnostic test for fast and accurate detection of chromosomal abnormalities in patients with developmental delay/intellectual disability (DD/ID), multiple congenital anomalies (MCA), and autism spectrum disorders (ASD). We present our experience with using CMA for postnatal and prenatal diagnosis in Estonian patients during 2009–2012. Since 2011, CMA is on the official service list of the Estonian Health Insurance Fund and is performed as the first-tier cytogenetic test for patients with DD/ID, MCA or ASD. A total of 1191 patients were analyzed, including postnatal (1072 [90%] patients and 59 [5%] family members) and prenatal referrals (60 [5%] fetuses). Abnormal results were reported in 298 (25%) patients, with a total of 351 findings (1–3 per individual): 147 (42%) deletions, 106 (30%) duplications, 89 (25%) long contiguous stretches of homozygosity (LCSH) events (>5 Mb), and nine (3%) aneuploidies. Of all findings, 143 (41%) were defined as pathogenic or likely pathogenic; for another 143 findings (41%), most of which were LCSH, the clinical significance remained unknown, while 61 (18%) reported findings can now be reclassified as benign or likely benign. Clinically relevant findings were detected in 126 (11%) patients. However, the proportion of variants of unknown clinical significance was quite high (41% of all findings). It seems that our ability to detect chromosomal abnormalities has far outpaced our ability to understand their role in disease. Thus, the interpretation of CMA findings remains a rather difficult task requiring a close collaboration between clinicians and cytogeneticists. PMID:24689080

  13. Association between FOXM1 and hedgehog signaling pathway in human cervical carcinoma by tissue microarray analysis

    PubMed Central

    Chen, Hong; Wang, Jingjing; Yang, Hong; Chen, Dan; Li, Panpan

    2016-01-01

    Forkhead box M1 (FOXM1) and hedgehog (Hh) signaling pathway are implicated in the formation and development of human tumors, including cervical cancer. Previous studies have indicated that FOXM1 may be a downstream target gene of the Hh signaling pathway, but their association in cervical cancer is largely unknown. In the present study, the expression of FOXM1 and Hh signaling molecules was evaluated by immunohistochemical analysis in a tissue microarray that contained 70 cervical cancer tissues and 10 normal cervical tissues. In addition, the association of these molecules with clinicopathological parameters, and the association between FOXM1 and various molecules involved in the Hh signaling pathway was investigated. The results indicated that FOXM1 and Hh signaling molecules were overexpressed in cervical cancer tissues. The protein expression levels of FOXM1, glioma-associated oncogene 1 (GLI1) and smoothened (SMO) correlated with the clinical stage of the tumors, while the protein expression levels of Sonic Hh (SHh), patched 1 (PTCH1) and GLI1 correlated with the pathological grade of the tumors. The expression levels of GLI1 were lower in tissues without lymph node metastasis than in tissues with lymph node metastasis. In addition, FOXM1 expression correlated with GLI1, SHh and PTCH1 expression in cancer tissues. These findings confirmed the participation of FOXM1 and the Hh signaling pathway in cervical cancer. Furthermore, the finding that FOXM1 may be a downstream target gene of the Hh signaling pathway in cervical cancer provides a potential novel diagnostic and therapeutic target for cervical cancer. PMID:27698840

  14. An examination of the regulatory mechanism of Pxdn mutation-induced eye disorders using microarray analysis

    PubMed Central

    YANG, YANG; XING, YIQIAO; LIANG, CHAOQUN; HU, LIYA; XU, FEI; MEI, QI

    2016-01-01

    The present study aimed to identify biomarkers for peroxidasin (Pxdn) mutation-induced eye disorders and study the underlying mechanisms involved in this process. The microarray dataset GSE49704 was used, which encompasses 4 mouse samples from embryos with Pxdn mutation and 4 samples from normal tissues. After data preprocessing, the differentially expressed genes (DEGs) between Pxdn mutation and normal tissues were identified using the t-test in the limma package, followed by functional enrichment analysis. The protein-protein interaction (PPI) network was constructed based on the STRING database, and the transcriptional regulatory (TR) network was established using the GeneCodis database. Subsequently, the overlapping DEGs with high degrees in two networks were identified, as well as the sub-network extracted from the TR network. In total, 121 (75 upregulated and 46 downregulated) DEGs were identified, and these DEGs play important roles in biological processes (BPs), including neuron development and differentiation. A PPI network containing 25 nodes such as actin, alpha 1, skeletal muscle (Acta1) and troponin C type 2 (fast) (Tnnc2), and a TR network including 120 nodes were built. By comparing the two networks, seven crucial genes which overlapped were identified, including cyclin-dependent kinase inhibitor 1B (Cdkn1b), Acta1 and troponin T type 3 (Tnnt3). In the sub-network, Cdkn1b was predicted as the target of miRNAs such as mmu-miR-24 and transcription factors (TFs) including forkhead box O4 (FOXO4) and activating enhancer binding protein 4 (AP4). Thus, we suggest that seven crucial genes, including Cdkn1b, Acta1 and Tnnt3, play important roles in the progression of eye disorders such as glaucoma. We suggest that Cdkn1b exert its effects via the inhibition of proliferation and is mediated by mmu-miR-24 and targeted by the TFs FOXO4 and AP4. PMID:27121343

  15. Microarray Cluster Analysis of Irradiated Growth Plate Zones Following Laser Microdissection

    SciTech Connect

    Damron, Timothy A. Zhang Mingliang; Pritchard, Meredith R.; Middleton, Frank A.; Horton, Jason A.; Margulies, Bryan M.; Strauss, Judith A.; Farnum, Cornelia E.; Spadaro, Joseph A.

    2009-07-01

    Purpose: Genes and pathways involved in early growth plate chondrocyte recovery after fractionated irradiation were sought as potential targets for selective radiorecovery modulation. Materials and Methods: Three groups of six 5-week male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days, totaling 17.5 Gy, and then were killed at 7, 11, and 16 days after the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibia using RAE230 2.0 GeneChip microarray, compared between zones and time points and subjected to functional pathway cluster analysis with real-time polymerase chain reaction to confirm selected results. Results: Each zone had a number of pathways showing enrichment after the pattern of hypothesized importance to growth plate recovery, yet few met the strictest criteria. The proliferative and hypertrophic zones showed both the greatest number of genes with a 10-fold right/left change at 7 days after initiation of irradiation and enrichment of the most functional pathways involved in bone, cartilage, matrix, or skeletal development. Six genes confirmed by real-time polymerase chain reaction to have early upregulation included insulin-like growth factor 2, procollagen type I alpha 2, matrix metallopeptidase 9, parathyroid hormone receptor 1, fibromodulin, and aggrecan 1. Conclusions: Nine overlapping pathways in the proliferative and hypertrophic zones (skeletal development, ossification, bone remodeling, cartilage development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part) may play key roles in early growth plate radiorecovery.

  16. Crowdsourcing for translational research: analysis of biomarker expression using cancer microarrays

    PubMed Central

    Lawson, Jonathan; Robinson-Vyas, Rupesh J; McQuillan, Janette P; Paterson, Andy; Christie, Sarah; Kidza-Griffiths, Matthew; McDuffus, Leigh-Anne; Moutasim, Karwan A; Shaw, Emily C; Kiltie, Anne E; Howat, William J; Hanby, Andrew M; Thomas, Gareth J; Smittenaar, Peter

    2017-01-01

    Background: Academic pathology suffers from an acute and growing lack of workforce resource. This especially impacts on translational elements of clinical trials, which can require detailed analysis of thousands of tissue samples. We tested whether crowdsourcing – enlisting help from the public – is a sufficiently accurate method to score such samples. Methods: We developed a novel online interface to train and test lay participants on cancer detection and immunohistochemistry scoring in tissue microarrays. Lay participants initially performed cancer detection on lung cancer images stained for CD8, and we measured how extending a basic tutorial by annotated example images and feedback-based training affected cancer detection accuracy. We then applied this tutorial to additional cancer types and immunohistochemistry markers – bladder/ki67, lung/EGFR, and oesophageal/CD8 – to establish accuracy compared with experts. Using this optimised tutorial, we then tested lay participants' accuracy on immunohistochemistry scoring of lung/EGFR and bladder/p53 samples. Results: We observed that for cancer detection, annotated example images and feedback-based training both improved accuracy compared with a basic tutorial only. Using this optimised tutorial, we demonstrate highly accurate (>0.90 area under curve) detection of cancer in samples stained with nuclear, cytoplasmic and membrane cell markers. We also observed high Spearman correlations between lay participants and experts for immunohistochemistry scoring (0.91 (0.78, 0.96) and 0.97 (0.91, 0.99) for lung/EGFR and bladder/p53 samples, respectively). Conclusions: These results establish crowdsourcing as a promising method to screen large data sets for biomarkers in cancer pathology research across a range of cancers and immunohistochemical stains. PMID:27959886

  17. Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

    SciTech Connect

    Bao, X.; Sinha, M. |; Liu, T.; Hong, C.; Luxon, B.A. |; Garofalo, R.P. ||; Casola, A. ||

    2008-04-25

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

  18. Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms.

    PubMed

    Franks, Ashley E; Nevin, Kelly P; Glaven, Richard H; Lovley, Derek R

    2010-04-01

    Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. Cells throughout anode biofilms of Geobacter sulfurreducens reduced the metabolic stains: 5-cyano-2,3-ditolyl tetrazolium chloride and Redox Green, suggesting metabolic activity throughout the biofilm. To compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, anode biofilms were encased in resin and sectioned into inner (0-20 microm from anode surface) and outer (30-60 microm) fractions. Transcriptional analysis revealed that, at a twofold threshold, 146 genes had significant (P<0.05) differences in transcript abundance between the inner and outer biofilm sections. Only 1 gene, GSU0093, a hypothetical ATP-binding cassette transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting lower metabolic rates. However, differences in transcript abundance were relatively low (microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.

  19. Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context?

    PubMed

    Benschop, Corina C G; Quaak, Frederike C A; Boon, Mathilde E; Sijen, Titia; Kuiper, Irene

    2012-03-01

    Forensic analysis of biological traces generally encompasses the investigation of both the person who contributed to the trace and the body site(s) from which the trace originates. For instance, for sexual assault cases, it can be beneficial to distinguish vaginal samples from skin or saliva samples. In this study, we explored the use of microbial flora to indicate vaginal origin. First, we explored the vaginal microbiome for a large set of clinical vaginal samples (n = 240) by next generation sequencing (n = 338,184 sequence reads) and found 1,619 different sequences. Next, we selected 389 candidate probes targeting genera or species and designed a microarray, with which we analysed a diverse set of samples; 43 DNA extracts from vaginal samples and 25 DNA extracts from samples from other body sites, including sites in close proximity of or in contact with the vagina. Finally, we used the microarray results and next generation sequencing dataset to assess the potential for a future approach that uses microbial markers to indicate vaginal origin. Since no candidate genera/species were found to positively identify all vaginal DNA extracts on their own, while excluding all non-vaginal DNA extracts, we deduce that a reliable statement about the cellular origin of a biological trace should be based on the detection of multiple species within various genera. Microarray analysis of a sample will then render a microbial flora pattern that is probably best analysed in a probabilistic approach.

  20. 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.

  1. Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres.

    PubMed

    Alonso, Alicia; Mahmood, Radma; Li, Shulan; Cheung, Fanny; Yoda, Kinya; Warburton, Peter E

    2003-10-15

    Human neocentromeres are fully functional centromeres that provide mitotic stability to rearranged chromosomes that have separated from endogenous centromeres. A disproportionate number of neocentromeres has been observed in certain regions such as chromosome 3q (n=6), 15q (n=9) and 13q32 (n=7), suggesting that these regions contain DNA sequences with a high propensity for neocentromere formation. Therefore, we have addressed the role of primary DNA sequence in neocentromere formation by asking whether multiple independent neocentromeres that were cytologically localized to chromosome 13q32 are in fact localized to the same underlying genomic DNA. Analysis of four independent 13q32 neocentromeres using simultaneous FISH with ordered YAC probes and immunofluorescence with antibodies to CENP-C have localized three neocentromeres to a distal approximately 7 Mb domain in chromosome 13q32, and one to an overlapping proximal domain of approximately 7 Mb. DNA was obtained from three of these neocentromeres by CENP-A chromatin immunoprecipitation (ChIP) and used to screen ordered BACs using both a slot-blotted BAC pool approach and a genomic microarray that contiguously spans 13q31.3-13q33.1. The CENP-A binding domains from each of these neocentromeres was identified to distinct genomic locations of approximately 130, 215 and 275 kb within an approximately 6.5 Mb region. Thus, the lack of coincidence of these neocentromeres to the same underlying DNA sequence refutes the idea of a DNA sequence based neocentromere 'hotspot' in 13q32 and further supports the sequence-independent epigenetic formation of human neocentromeres. The screening of genomic microarrays with ChIP DNA provides a powerful method to identify mammalian DNA sequences associated with particular functional chromatin states.

  2. Comparative analysis of microarray data in Arabidopsis transcriptome during compatible interactions with plant viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To analyze transcriptome response to virus infection, we have assembled currently available microarray data on changes in gene expression levels in compatible Arabidopsis-virus interactions. We used the mean r (Pearson’s correlation coefficient) for neighboring pairs to estimate pairwise local simil...

  3. Microarray analysis of gene expression and diapause in Glassy-winged Sharpshooter (Homalodisca vitripennis: Hemiptera: Cicadellidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The condition of diapause in the glassy-winged sharpshooter, GWSS, Homalodisca vitripennis, is poorly understood. Diapause is better known from other, non hemipteran insects. We used oligonucleotide microarrays to address the specificities of transcriptional responses of adult female GWSS, which wer...

  4. Microarray as a First Genetic Test in Global Developmental Delay: A Cost-Effectiveness Analysis

    ERIC Educational Resources Information Center

    Trakadis, Yannis; Shevell, Michael

    2011-01-01

    Aim: Microarray technology has a significantly higher clinical yield than karyotyping in individuals with global developmental delay (GDD). Despite this, it has not yet been routinely implemented as a screening test owing to the perception that this approach is more expensive. We aimed to evaluate the effect that replacing karyotype with…

  5. DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells

    SciTech Connect

    Sadikovic, Bekim; Andrews, Joseph; Rodenhiser, David I.

    2007-12-15

    Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes.

  6. Microarray analysis of microRNA expression in the developing mammalian brain

    PubMed Central

    Miska, Eric A; Alvarez-Saavedra, Ezequiel; Townsend, Matthew; Yoshii, Akira; Šestan, Nenad; Rakic, Pasko; Constantine-Paton, Martha; Horvitz, H Robert

    2004-01-01

    Background MicroRNAs are a large new class of tiny regulatory RNAs found in nematodes, plants, insects and mammals. MicroRNAs are thought to act as post-transcriptional modulators of gene expression. In invertebrates microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death and fat metabolism. Little is known about the roles of microRNAs in mammals. Results We isolated 18-26 nucleotide RNAs from developing rat and monkey brains. From the sequences of these RNAs and the sequences of the rat and human genomes we determined which of these small RNAs are likely to have derived from stem-loop precursors typical of microRNAs. Next, we developed a microarray technology suitable for detecting microRNAs and printed a microRNA microarray representing 138 mammalian microRNAs corresponding to the sequences of the microRNAs we cloned as well as to other known microRNAs. We used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain. Conclusion We describe a microarray technology that can be used to analyze the expression of microRNAs and of other small RNAs. MicroRNA microarrays offer a new tool that should facilitate studies of the biological roles of microRNAs. We used this method to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs. PMID:15345052

  7. The Microarray Revolution: Perspectives from Educators

    ERIC Educational Resources Information Center

    Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

    2004-01-01

    In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research,…

  8. Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki)

    PubMed Central

    2013-01-01

    Background The eastern mosquitofish (Gambusia holbrooki) has the potential to become a bioindicator organism of endocrine disrupting chemicals (EDCs) due to its androgen-driven secondary sexual characteristics. However, the lack of molecular information on G. holbrooki hinders its use as a bioindicator coupled with biomarker data. While traditional gene-by-gene approaches provide insight for biomarker development, a holistic analysis would provide more rapid and expansive determination of potential biomarkers. The objective of this study was to develop and utilize a mosquitofish microarray to determine potential biomarkers of subchronic androgen exposure. To achieve this objective, two specific aims were developed: 1) Sequence a G. holbrooki cDNA library, and 2) Use microarray analysis to determine genes that are differentially regulated by subchronic androgen exposure in hepatic tissues of 17β-trenbolone (TB) exposed adult female G. holbrooki. Results A normalized library of multiple organs of male and female G. holbrooki was prepared and sequenced by the Illumina GA IIx and Roche 454 XLR70. Over 30,000 genes with e-value ≤ 10-4 were annotated and 14,758 of these genes were selected for inclusion on the microarray. Hepatic microarray analysis of adult female G. holbrooki exposed to the vehicle control or 1 μg/L of TB (a potent anabolic androgen) revealed 229 genes upregulated and 279 downregulated by TB (one-way ANOVA, p < 0.05, FDR α = 0.05, fold change > 1.5 and < −1.5). Fifteen gene ontology biological processes were enriched by TB exposure (Fisher’s Exact Test, p < 0.05). The expression levels of 17β-hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student’s t-test, p < 0.05). Conclusions Coupling microarray data with phenotypic changes driven by androgen exposure in mosquitofish is key for developing this organism into a bioindicator for

  9. Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.

    PubMed

    Trask, Heidi W; Cowper-Sal-lari, Richard; Sartor, Maureen A; Gui, Jiang; Heath, Catherine V; Renuka, Janhavi; Higgins, Azara-Jane; Andrews, Peter; Korc, Murray; Moore, Jason H; Tomlinson, Craig R

    2009-10-01

    With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.

  10. Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services.

    PubMed

    Roberts, Jennifer L; Hovanes, Karine; Dasouki, Majed; Manzardo, Ann M; Butler, Merlin G

    2014-02-01

    Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105 K and 180K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009-2012). Of the 215 patients [140 males and 75 females (male/female ratio=1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n=20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n=8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group.

  11. Chromosomal Microarray Analysis of Consecutive Individuals with Autism Spectrum Disorders or Learning Disability Presenting for Genetic Services

    PubMed Central

    Roberts, Jennifer L.; Hovanes, Karine; Dasouki, Majed; Manzardo, Ann M.; Butler, Merlin G.

    2015-01-01

    Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105K and 180K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009–2012). Of the 215 patients [140 males and 75 females (male/female ratio = 1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n = 20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n = 8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group. PMID:24188901

  12. Integrative RNA-seq and microarray data analysis reveals GC content and gene length biases in the psoriasis transcriptome

    PubMed Central

    Xing, Xianying; Voorhees, John J.; Elder, James T.; Johnston, Andrew; Gudjonsson, Johann E.

    2014-01-01

    Gene expression profiling of psoriasis has driven research advances and may soon provide the basis for clinical applications. For expression profiling studies, RNA-seq is now a competitive technology, but RNA-seq results may differ from those obtained by microarray. We therefore compared findings obtained by RNA-seq with those from eight microarray studies of psoriasis. RNA-seq and microarray datasets identified similar numbers of differentially expressed genes (DEGs), with certain genes uniquely identified by each technology. Correspondence between platforms and the balance of increased to decreased DEGs was influenced by mRNA abundance, GC content, and gene length. Weakly expressed genes, genes with low GC content, and long genes were all biased toward decreased expression in psoriasis lesions. The strength of these trends differed among array datasets, most likely due to variations in RNA quality. Gene length bias was by far the strongest trend and was evident in all datasets regardless of the expression profiling technology. The effect was due to differences between lesional and uninvolved skin with respect to the genome-wide correlation between gene length and gene expression, which was consistently more negative in psoriasis lesions. These findings demonstrate the complementary nature of RNA-seq and microarray technology and show that integrative analysis of both data types can provide a richer view of the transcriptome than strict reliance on a single method alone. Our results also highlight factors affecting correspondence between technologies, and we have established that gene length is a major determinant of differential expression in psoriasis lesions. PMID:24844236

  13. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed.

  14. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination.

    PubMed

    Li, Lu; Wang, Xianwei; Zhang, Xiaoli; Wang, Jinxing; Jin, Wenrui

    2015-01-07

    We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3×10(-16) mol L(-1). The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of cDNAs corresponding to beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P2 mRNAs in single human breast cancer cells and 5 random synthetic DNAts are simultaneously quantified to examine the SMA and SMA-based single-cell multiple gene expression analysis.

  15. Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research*

    PubMed Central

    Pedersen, Henriette L.; Fangel, Jonatan U.; McCleary, Barry; Ruzanski, Christian; Rydahl, Maja G.; Ralet, Marie-Christine; Farkas, Vladimir; von Schantz, Laura; Marcus, Susan E.; Andersen, Mathias C. F.; Field, Rob; Ohlin, Mats; Knox, J. Paul; Clausen, Mads H.; Willats, William G. T.

    2012-01-01

    Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes. PMID:22988248

  16. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    PubMed

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars.

  17. Microarrays for Undergraduate Classes

    ERIC Educational Resources Information Center

    Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

    2006-01-01

    A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

  18. Personalised proteome analysis by means of protein microarrays made from individual patient samples

    PubMed Central

    Syafrizayanti; Lueong, Smiths S.; Di, Cuixia; Schaefer, Jonas V.; Plückthun, Andreas; Hoheisel, Jörg D.

    2017-01-01

    DNA sequencing has advanced to a state that permits studying the genomes of individual patients as nearly a matter of routine. Towards analysing a tissue’s protein content in a similar manner, we established a method for the production of microarrays that represent full-length proteins as they are encoded in individual specimens, exhibiting the particular variations, such as mutations or splice variations, present in these samples. From total RNA isolates, each transcript is copied to a specific location on the array by an on-chip polymerase elongation reaction, followed by in situ cell-free transcription and translation. These microarrays permit parallel analyses of variations in protein structure and interaction that are specific to particular samples. PMID:28045055

  19. Estimating equation–based causality analysis with application to microarray time series data

    PubMed Central

    Hu, Jianhua; Hu, Feifang

    2009-01-01

    Microarray time-course data can be used to explore interactions among genes and infer gene network. The crucial step in constructing gene network is to develop an appropriate causality test. In this regard, the expression profile of each gene can be treated as a time series. A typical existing method establishes the Granger causality based on Wald type of test, which relies on the homoscedastic normality assumption of the data distribution. However, this assumption can be seriously violated in real microarray experiments and thus may lead to inconsistent test results and false scientific conclusions. To overcome the drawback, we propose an estimating equation–based method which is robust to both heteroscedasticity and nonnormality of the gene expression data. In fact, it only requires the residuals to be uncorrelated. We will use simulation studies and a real-data example to demonstrate the applicability of the proposed method. PMID:19329818

  20. Microarray gene expression analysis of the human airway in patients exposed to sulfur mustard.

    PubMed

    Najafi, Ali; Masoudi-Nejad, Ali; Imani Fooladi, Abbas Ali; Ghanei, Mostafa; Nourani, Mohamad Reza

    2014-08-01

    There is much data about the acute effects of sulfur mustard gas on humans, animals and cells. But less is known regarding the molecular basics of chronic complications in humans. Basically, mustard gas, as an alkylating agent, causes several chronic problems in the eyes, skin and more importantly in the pulmonary system which is the main cause of death. Although recent proteomic research has been carried out on bronchoalveolar lavage (BAL) and serum, but high-throughput transcriptomics have not yet been applied to chronic airway remodeling. This is the first cDNA-microarray report on the chronic human mustard lung disease, 25 years after exposure during the Iran-Iraq war. Microarray transcriptional profiling indicated that a total of 122 genes were significantly dysregulated in tissues located in the airway of patients. These genes are associated with the extracellular matrix components, apoptosis, stress response, inflammation and mucus secretion.

  1. DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae.

    PubMed

    Yasokawa, Daisuke; Murata, Satomi; Iwahashi, Yumiko; Kitagawa, Emiko; Kishi, Katsuyuki; Okumura, Yukihiro; Iwahashi, Hitoshi

    2010-05-01

    Zinc pyrithione has been used in anti-dandruff shampoos and in anti-fouling paint on ships. However, little is known of its mode of action. We characterized the effects of sub-lethal concentrations of zinc pyrithione (Zpt) on Saccharomyces cerevisiae using DNA microarrays. The majority of the strongly upregulated genes are related to iron transport, and many of the strongly downregulated genes are related to the biosynthesis of cytochrome (heme). These data suggest that Zpt induces severe iron starvation. To confirm the DNA microarray data, we supplemented cultures containing Zpt with iron, and the growth of the yeast was restored significantly. From these results, we propose that the principal toxicity of zinc pyrithione arises from iron starvation.

  2. 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

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

    PubMed

    Thissen, James B; McLoughlin, Kevin; Gardner, Shea; Gu, Pauline; Mabery, Shalini; Slezak, Tom; Jaing, Crystal

    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 1h. The LLMDA was 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 1h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1h 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.

  4. Quantitative methods for genome-scale analysis of in situ hybridization and correlation with microarray data

    PubMed Central

    Lee, Chang-Kyu; Sunkin, Susan M; Kuan, Chihchau; Thompson, Carol L; Pathak, Sayan; Ng, Lydia; Lau, Chris; Fischer, Shanna; Mortrud, Marty; Slaughterbeck, Cliff; Jones, Allan; Lein, Ed; Hawrylycz, Michael

    2008-01-01

    With the emergence of genome-wide colorimetric in situ hybridization (ISH) data sets such as the Allen Brain Atlas, it is important to understand the relationship between this gene expression modality and those derived from more quantitative based technologies. This study introduces a novel method for standardized relative quantification of colorimetric ISH signal that enables a large-scale cross-platform expression level comparison of ISH with two publicly available microarray brain data sources. PMID:18234097

  5. A New Modified Histogram Matching Normalization for Time Series Microarray Analysis.

    PubMed

    Astola, Laura; Molenaar, Jaap

    2014-07-01

    Microarray data is often utilized in inferring regulatory networks. Quantile normalization (QN) is a popular method to reduce array-to-array variation. We show that in the context of time series measurements QN may not be the best choice for this task, especially not if the inference is based on continuous time ODE model. We propose an alternative normalization method that is better suited for network inference from time series data.

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

    SciTech Connect

    Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; Gu, Pauline; Mabery, Shalini; Slezak, Tom; Jaing, Crystal

    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 was 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.

  7. Microarray analysis of gene expression in olive flounder liver infected with viral haemorrhagic septicaemia virus (VHSV).

    PubMed

    Cho, Hyun Kook; Kim, Julan; Moon, Ji Young; Nam, Bo-Hye; Kim, Young-Ok; Kim, Woo-Jin; Park, Jung Youn; An, Cheul Min; Cheong, Jaehun; Kong, Hee Jeong

    2016-02-01

    The most fatal viral pathogen in olive flounder Paralichthys olivaceus, is viral hemorrhagic septicemia virus, which afflicts over 48 species of freshwater and marine fish. Here, we performed gene expression profiling on transcripts isolated from VHSV-infected olive flounder livers using a 13 K cDNA microarray chip. A total of 1832 and 1647 genes were upregulated and down-regulated over two-fold, respectively, after infection. A variety of immune-related genes showing significant changes in gene expression were identified in upregulated genes through gene ontology annotation. These genes were grouped into categories such as antibacterial peptide, antigen-recognition and adhesion molecules, apoptosis, cytokine-related pathway, immune system, stress response, and transcription factor and regulatory factors. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to the microarray data. In conclusion, these results may be useful for the identification of specific genes or for the diagnosis of VHSV infection in flounder.

  8. Borrowing information across genes and experiments for improved error variance estimation in microarray data analysis.

    PubMed

    Ji, Tieming; Liu, Peng; Nettleton, Dan

    2012-01-01

    Statistical inference for microarray experiments usually involves the estimation of error variance for each gene. Because the sample size available for each gene is often low, the usual unbiased estimator of the error variance can be unreliable. Shrinkage methods, including empirical Bayes approaches that borrow information across genes to produce more stable estimates, have been developed in recent years. Because the same microarray platform is often used for at least several experiments to study similar biological systems, there is an opportunity to improve variance estimation further by borrowing information not only across genes but also across experiments. We propose a lognormal model for error variances that involves random gene effects and random experiment effects. Based on the model, we develop an empirical Bayes estimator of the error variance for each combination of gene and experiment and call this estimator BAGE because information is Borrowed Across Genes and Experiments. A permutation strategy is used to make inference about the differential expression status of each gene. Simulation studies with data generated from different probability models and real microarray data show that our method outperforms existing approaches.

  9. Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis

    PubMed Central

    Palmer, Ella L; Miller, Andrew D; Freeman, Tom C

    2006-01-01

    Background Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a powerful new approach for performing high throughput screens of gene function. An important application of cell-based microarrays is in screening for proteins that modulate gene networks. To this end, cells are grown over the surface of arrays of RNAi or expression reagents. Cells growing in the immediate vicinity of the arrayed reagents are transfected and the arrays can then be scanned for cells showing localised changes in function. Here we describe the construction of a large-scale microarray using expression plasmids containing human genes, its use in screening for genes that induce apoptosis when over-expressed and the characterisation of a number of these genes by following the transcriptional response of cell cultures during their induction of apoptosis. Results High-density cell-based arrays were successfully fabricated using 1,959 un-tagged open reading frames (ORFs) taken from the Mammalian Gene Collection (MGC) in mammalian expression vectors. The arrays were then used to screen for genes inducing apoptosis in Human Embryonic Kidney (HEK293T) cells. Using this approach, 10 genes were clearly identified and confirmed to induce apoptosis. Some of these genes have previously been linked to apoptosis, others not. The mechanism of action of three of the 10 genes were then characterised further by following the transcriptional events associated with apoptosis induction using expression profiling microarrays. This data demonstrates a clear pro-apoptotic transcriptional response in cells undergoing apoptosis and also suggests the use of common apoptotic pathways regardless of the nature of the over-expressed protein triggering cell death. Conclusion This study reports the design and use of the first truly large-scale cell-based microarrays for over-expression studies. Ten genes were confirmed to induce apoptosis, some of which were not previously known to possess this

  10. Microarray analysis of CA1 pyramidal neurons in a mouse model of tauopathy reveals progressive synaptic dysfunction

    PubMed Central

    Alldred, Melissa J.; Duff, Karen E.; Ginsberg, Stephen D.

    2011-01-01

    The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11-14 months. Statistical analysis revealed ∼8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin (Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7-8 months of age indicated only a few alterations compared to the 11-14 month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD. PMID:22079237

  11. Transcriptomic response of murine liver to severe injury and hemorrhagic shock: a dual-platform microarray analysis

    PubMed Central

    Edmonds, Rebecca D.; Lagoa, Claudio; Dutta-Moscato, Joyeeta; Yang, Yawching; Fink, Mitchell P.; Levy, Ryan M.; Prince, Jose M.; Kaczorowski, David J.; Tseng, George C.; Billiar, Timothy R.

    2011-01-01

    Trauma-hemorrhagic shock (HS/T) is a complex process that elicits numerous molecular pathways. We hypothesized that a dual-platform microarray analysis of the liver, an organ that integrates immunology and metabolism, would reveal key pathways engaged following HS/T. C57BL/6 mice were divided into five groups (n = 4/group), anesthetized, and surgically treated to simulate a time course and trauma severity model: 1) nonmanipulated animals, 2) minor trauma, 3) 1.5 h of hemorrhagic shock and severe trauma (HS/T), 4) 1.5 h HS/T followed by 1 h resuscitation (HS/T+1.0R), 5) 1.5 h HS/T followed by 4.5 h resuscitation (HS/T+4.5R). Liver RNA was hybridized to CodeLink and Affymetrix mouse whole genome microarray chips. Common genes with a cross-platform correlation >0.6 (2,353 genes in total) were clustered using k-means clustering, and clusters were analyzed using Ingenuity Pathways Analysis. Genes involved in the stress response and immunoregulation were upregulated early and remained upregulated throughout the course of the experiment. Genes involved in cell death and inflammatory pathways were upregulated in a linear fashion with elapsed time and in severe injury compared with minor trauma. Three of the six clusters contained genes involved in metabolic function; these were downregulated with elapsed time. Transcripts involved in amino acid metabolism as well as signaling pathways associated with glucocorticoid receptors, IL-6, IL-10, and the acute phase response were elevated in a severity-dependent manner. This is the first study to examine the postinjury response using dual-platform microarray analysis, revealing responses that may enable novel therapies or diagnostics. PMID:21828244

  12. Microarray-based analysis of gene expression in lycopersicon esculentum seedling roots in response to cadmium, chromium, mercury, and lead.

    PubMed

    Hou, Jing; Liu, Xinhui; Wang, Juan; Zhao, Shengnan; Cui, Baoshan

    2015-02-03

    The effects of heavy metals in agricultural soils have received special attention due to their potential for accumulation in crops, which can affect species at all trophic levels. Therefore, there is a critical need for reliable bioassays for assessing risk levels due to heavy metals in agricultural soil. In the present study, we used microarrays to investigate changes in gene expression of Lycopersicon esculentum in response to Cd-, Cr-, Hg-, or Pb-spiked soil. Exposure to (1)/10 median lethal concentrations (LC50) of Cd, Cr, Hg, or Pb for 7 days resulted in expression changes in 29 Cd-specific, 58 Cr-specific, 192 Hg-specific and 864 Pb-specific genes as determined by microarray analysis, whereas conventional morphological and physiological bioassays did not reveal any toxicant stresses. Hierarchical clustering analysis showed that the characteristic gene expression profiles induced by Cd, Cr, Hg, and Pb were distinct from not only the control but also one another. Furthermore, a total of three genes related to "ion transport" for Cd, 14 genes related to "external encapsulating structure organization", "reproductive developmental process", "lipid metabolic process" and "response to stimulus" for Cr, 11 genes related to "cellular metabolic process" and "cellular response to stimulus" for Hg, 78 genes related to 20 biological processes (e.g., DNA metabolic process, monosaccharide catabolic process, cell division) for Pb were identified and selected as their potential biomarkers. These findings demonstrated that microarray-based analysis of Lycopersicon esculentum was a sensitive tool for the early detection of potential toxicity of heavy metals in agricultural soil, as well as an effective tool for identifying the heavy metal-specific genes, which should be useful for assessing risk levels due to heavy metals in agricultural soil.

  13. Gene microarray analysis of lncRNA and mRNA expression profiles in patients with hypopharyngeal squamous cell carcinoma

    PubMed Central

    Zhou, Jieyu; Li, Wenming; Jin, Tong; Xiang, Xuan; Li, Maocai; Wang, Juan; Li, Guojun; Pan, Xinliang; Lei, Dapeng

    2015-01-01

    Background: Studies have shown that long noncoding RNAs (lncRNAs) are involved in the development and progression of many types of cancer. However, the mechanisms by which lncRNAs influence development and progression of hypopharyngeal squamous cell carcinoma (HSCC) are unclear. Method: We investigated differences in lncRNA and mRNA expression profiles between 3 pairs of HSCC tissues and adjacent nontumor tissues by microarray analysis. Results: In HSCC tissues, 1299 lncRNAs were significantly upregulated (n=669) or downregulated (n=630) compared to levels in adjacent nontumor tissues. Moreover, 1432 mRNAs were significantly upregulated (n=684) or downregulated (n=748) in HSCC tissues. We randomly selected 2 differentially expressed lncRNAs (AB209630, AB019562) and 2 differentially expressed mRNAs (SPP1, TJP2) for confirmation of microarray results using qRT-PCR. The qRT-PCR results matched well with the microarray data. The differentially expressed lncRNAs and mRNAs were distributed on each of the chromosomes, including the X and Y chromosomes. Pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 48 cellular pathways that may be associated with HSCC development. GO analysis revealed that 593 mRNAs involved in biological processes, 50 mRNAs involved in cellular components, and 46 mRNAs involved in molecular functions were upregulated in the carcinomas; 280 mRNAs involved in biological processes, 58 mRNAs involved in cellular components, and 71 mRNAs involved in molecular functions were downregulated in the carcinomas. In addition, 8 enhancer-like lncRNAs and 21 intergenic lncRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Conclusion: These findings provide insight into the mechanisms underlying HSCC tumorigenesis and will facilitate identification of new therapeutic targets and diagnostic biomarkers for this disease. PMID:26131061

  14. A combined analysis of microarray gene expression studies of the human prefrontal cortex identifies genes implicated in schizophrenia.

    PubMed

    Pérez-Santiago, Josué; Diez-Alarcia, Rebeca; Callado, Luis F; Zhang, Jin X; Chana, Gursharan; White, Cory H; Glatt, Stephen J; Tsuang, Ming T; Everall, Ian P; Meana, J Javier; Woelk, Christopher H

    2012-11-01

    Small cohort sizes and modest levels of gene expression changes in brain tissue have plagued the statistical approaches employed in microarray studies investigating the mechanism of schizophrenia. To combat these problems a combined analysis of six prior microarray studies was performed to facilitate the robust statistical analysis of gene expression data from the dorsolateral prefrontal cortex of 107 patients with schizophrenia and 118 healthy subjects. Multivariate permutation tests identified 144 genes that were differentially expressed between schizophrenia and control groups. Seventy of these genes were identified as differentially expressed in at least one component microarray study but none of these individual studies had the power to identify the remaining 74 genes, demonstrating the utility of a combined approach. Gene ontology terms and biological pathways that were significantly enriched for differentially expressed genes were related to neuronal cell-cell signaling, mesenchymal induction, and mitogen-activated protein kinase signaling, which have all previously been associated with the etiopathogenesis of schizophrenia. The differential expression of BAG3, C4B, EGR1, MT1X, NEUROD6, SST and S100A8 was confirmed by real-time quantitative PCR in an independent cohort using postmortem human prefrontal cortex samples. Comparison of gene expression between schizophrenic subjects with and without detectable levels of antipsychotics in their blood suggests that the modulation of MT1X and S100A8 may be the result of drug exposure. In conclusion, this combined analysis has resulted in a statistically robust identification of genes whose dysregulation may contribute to the mechanism of schizophrenia.

  15. The epidermal growth factor receptor is frequently overexpressed in penile squamous cell carcinomas: a tissue microarray and digital image analysis study of 112 cases.

    PubMed

    Chaux, Alcides; Munari, Enrico; Katz, Betina; Sharma, Rajni; Lecksell, Kristen; Cubilla, Antonio L; Burnett, Arthur L; Netto, George J

    2013-12-01

    Disseminated penile cancer is usually treated with chemotherapy. However, response rates are far from acceptable. Recently, anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have shown to be clinically useful in penile carcinomas. Nevertheless, only a few cases of penile carcinomas have been evaluated for EGFR expression. In this study, we assessed the immunohistochemical expression of EGFR in 112 patients with penile squamous cell carcinoma. We built 4 tissue microarrays and evaluated EGFR expression using a monoclonal mouse anti-EGFR antibody. For digital image analysis, we used the open-source software ImageJ version 1.47 (NIH, Bethesda, MD) along with the immunomembrane plug-in. Membranous EGFR expression was evaluated, taking into account staining completeness (0-10 points) and staining intensity (0-10 points) for a combined score (0-20 points). We classified the cases as follows: negative EGFR expression, 0 to 3 points; low EGFR expression, 4 to 8 points; and high EGFR expression, 9 to 20 points. The distribution of EGFR immunohistochemical expression was as follows: 13 cases (12%) were EGFR negative, 49 cases (44%) had low EGFR expression, and 50 cases (44%) had high EGFR expression. EGFR expression was not associated with histologic subtype (P = .47), histologic grade (P = .77), or human papillomavirus status (P = .14). In conclusion, immunohistochemical EGFR expression appears to be a common feature of penile carcinomas, independently of histologic subtype, histologic grade, and human papillomavirus presence. Whether or not EGFR expression is associated with EGFR gene mutation or if it can be used to predict response to therapy in patients with disseminated penile cancer should be evaluated in future studies.

  16. BR 07-1 DEVELOPMENT OF THE CELL MICROARRAY FOR HIGH-THROUGHPUT ANALYSIS OF GUT MICROBIOTA.

    PubMed

    Hong, Seong-Tshool

    2016-09-01

    The human intestine contains a massive and complex microbial community called gut microbiota. A typical human carries 100 trillion microbes in his/her body which is 10 times greater than the number of their host cells, i.e. whole number of human cells. A combined microbial genome constituting gut microbiota is well excess our own human genome. The microbial composition of gut microbiotata and its role on diseases became a booming area of research, presenting a new paradigm of opportunities for modern medicines. Recent evidences showed that gut microbiota acts as a very important determining factor for the development of almost all complex diseases such as primary hypertension, obesity, depression, diabetes, autism, asthma, bowl diseases, rheumatic arthritis, systemic lupus erythematosus, Crohn's disease, Parkinson's disease, Alzheimer's disease, epilepsy, schizophrenia, etc. In spite of the significant role of gut microbiota in the development of complex diseases, the elucidation of the mechanistic pathway on the development of complex diseases by gut microbiota is not moving forward as expected. Current methods to identify alteration of gut microbiota in patients and healthy controls are basically based on the metagenomic sequencings of DNA samples extracted from feces by using next-generation sequencing machines. Although the metagenomic sequencing approaches proved association of gut microbiota with various complex diseases, those methods failed to accurately pinpoint the etiological agents in gut microbiota for complex diseases. The metagenomic sequencing approaches are not only difficult to identify the etiological agent of complex diseases at species level but also difficult to use, requiring complex bioinformatic analyses, and expensive. To overcome the current challenges in analysis of gut microbiota, we developed a novel cell microarray to analyze the constituent microbial organisms of gut microbiota very accurately and fast by using a drop of blood. The

  17. Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays.

    PubMed

    Zhou, Xichun; Zhou, Jizhong

    2006-03-01

    We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.

  18. Functional microarray analysis of nitrogen and carbon cycling genes across an Antarctic latitudinal transect.

    PubMed

    Yergeau, Etienne; Kang, Sanghoon; He, Zhili; Zhou, Jizhong; Kowalchuk, George A

    2007-06-01

    Soil-borne microbial communities were examined via a functional gene microarray approach across a southern polar latitudinal gradient to gain insight into the environmental factors steering soil N- and C-cycling in terrestrial Antarctic ecosystems. The abundance and diversity of functional gene families were studied for soil-borne microbial communities inhabiting a range of environments from 51 degrees S (cool temperate-Falkland Islands) to 72 degrees S (cold rock desert-Coal Nunatak). The recently designed functional gene array used contains 24,243 oligonucleotide probes and covers >10,000 genes in >150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance and organic contaminant degradation (He et al. 2007). The detected N- and C-cycle genes were significantly different across different sampling locations and vegetation types. A number of significant trends were observed regarding the distribution of key gene families across the environments examined. For example, the relative detection of cellulose degradation genes was correlated with temperature, and microbial C-fixation genes were more present in plots principally lacking vegetation. With respect to the N-cycle, denitrification genes were linked to higher soil temperatures, and N2-fixation genes were linked to plots mainly vegetated by lichens. These microarray-based results were confirmed for a number of gene families using specific real-time PCR, enzymatic assays and process rate measurements. The results presented demonstrate the utility of an integrated functional gene microarray approach in detecting shifts in functional community properties in environmental samples and provide insight into the forces driving important processes of terrestrial Antarctic nutrient cycling.

  19. Telomerase as a prognostic marker in breast cancer: high-throughput tissue microarray analysis of hTERT and hTR.

    PubMed

    Poremba, Christopher; Heine, Bernhard; Diallo, Raihanatou; Heinecke, Achim; Wai, Daniel; Schaefer, Karl-Ludwig; Braun, Yvonne; Schuck, Andreas; Lanvers, Claudia; Bànkfalvi, Agnes; Kneif, Sören; Torhorst, Joachim; Zuber, Markus; Köchli, Ossi R; Mross, Frank; Dieterich, Holger; Sauter, Guido; Stein, Harald; Fogt, Franz; Boecker, Werner

    2002-10-01

    Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin-embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse-transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti-telomerase antibody Ab-2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi-quantitatively and graded (scores 1-4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour-specific survival (in months), patient's age and others, were available for statistical analysis. A statistically significant correlation was found between tumour-specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh-frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival

  20. Spectral Analysis of Breast Cancer on Tissue Microarrays: Seeing Beyond Morphology

    DTIC Science & Technology

    2004-05-01

    Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and should not be construed as an...of Breast Cancer on Tissue Microarrays: DAMD17-02-1-0634 Seeing Beyond Morphology 6. AUTHOR( S ) David L. Rimm, M.D., Ph.D. 7. PERFORMING ORGANIZATION...NAME( S ) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Yale University School of Medicine REPORT NUMBER New Haven, CT 06520-8047 E-Mail: david. rimm@yale

  1. Network Theory Analysis of Antibody-Antigen Reactivity Data: The Immune Trees at Birth and Adulthood

    PubMed Central

    Bransburg-Zabary, Sharron; Merbl, Yifat; Quintana, Francisco J.; Tauber, Alfred I.; Cohen, Irun R.; Ben-Jacob, Eshel

    2011-01-01

    Motivation New antigen microarray technology enables parallel recording of antibody reactivities with hundreds of antigens. Such data affords system level analysis of the immune system's organization using methods and approaches from network theory. Here we measured the reactivity of 290 antigens (for both the IgG and IgM isotypes) of 10 healthy mothers and their term newborns. We constructed antigen correlation networks (or immune networks) whose nodes are the antigens and the edges are the antigen-antigen reactivity correlations, and we also computed their corresponding minimum spanning trees (MST) – maximal information reduced sub-graphs. We quantify the network organization (topology) in terms of the network theory divergence rate measure and rank the antigen importance in the full antigen correlation networks by the eigen-value centrality measure. This analysis makes possible the characterization and comparison of the IgG and IgM immune networks at birth (newborns) and adulthood (mothers) in terms of topology and node importance. Results Comparison of the immune network topology at birth and adulthood revealed partial conservation of the IgG immune network topology, and significant reorganization of the IgM immune networks. Inspection of the antigen importance revealed some dominant (in terms of high centrality) antigens in the IgG and IgM networks at birth, which retain their importance at adulthood. PMID:21408156

  2. Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica.

    PubMed

    Lindsey, Rebecca L; Frye, Jonathan G; Fedorka-Cray, Paula J; Meinersmann, Richard J

    2011-10-01

    In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.

  3. The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal.

    PubMed

    Wang, C; Francis, R; Harirchian, S; Batlle, D; Mayhew, B; Bassett, M; Rainey, W E; Pestell, R G

    2000-11-01

    Angiotensin II (AII) binds to specific G-protein coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. The H295R human adrenocortical cell line, which expresses AII receptors predominantly of the AT1 subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a precisely coordinated induction of a variety of genes. As the human genome sequencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic responses. One high throughput method, the gridded cDNA microarray has been developed in which immobilised DNA samples are hybridized on glass slides for the identification of global genomic responses. For this purpose high precision robotic microarrayers have been developed at AECOM. The cyclin D1 gene, which encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was induced by AII in H295R cells. Abundance of the cyclin D1 gene is rate-limiting in G1 phase progression of the cell-cycle in a variety of cell types. AII induced cyclin D1 promoter activity through a c-Fos and c-Jun binding sequence at -954 bp. Theabundance of c-Fos within this complex was increased by AII treatment. Analysis of AII signaling in adrenal cells by cDNA microarray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXPMC2). The cDNA for XPMC2 was previously shown to rescue mitotic catastrophe in mutant S. Pombe defective in cdc2 kinase function. Further studies are required to determine the requirement for cyclin D1 and XPMC2H in AII-induced cell-cycle progression and cellular proliferation in the adrenal.

  4. Symbolic Data Analysis to Defy Low Signal-to-Noise Ratio in Microarray Data for Breast Cancer Prognosis

    PubMed Central

    Le Lann, Marie-Veronique; Kempowsky, Tatiana; Dalenc, Florence; Aguilar-Martin, Joseph; Favre, Gilles

    2013-01-01

    Abstract Microarray profiling has recently generated the hope to gain new insights into breast cancer biology and thereby improve the performance of current prognostic tools. However, it also poses several serious challenges to classical data analysis techniques related to the characteristics of resulting data, mainly high dimensionality and low signal-to-noise ratio. Despite the tremendous research work performed to handle the first challenge in the feature selection framework, very little attention has been directed to address the second one. We propose in this article to address both issues simultaneously based on symbolic data analysis capabilities in order to derive more accurate genetic marker–based prognostic models. In particular, interval data representation is employed to model various uncertainties in microarray measurements. A recent feature selection algorithm that handles symbolic interval data is used then to derive a genetic signature. The predictive value of the derived signature is then assessed by following a rigorous experimental setup and compared with existing prognostic approaches in terms of predictive performance and estimated survival probability. It is shown that the derived signature (GenSym) performs significantly better than other prognostic models, including the 70-gene signature, St. Gallen, and National Institutes of Health criteria. PMID:23899014

  5. Symbolic data analysis to defy low signal-to-noise ratio in microarray data for breast cancer prognosis.

    PubMed

    Hedjazi, Lyamine; Le Lann, Marie-Veronique; Kempowsky, Tatiana; Dalenc, Florence; Aguilar-Martin, Joseph; Favre, Gilles

    2013-08-01

    Microarray profiling has recently generated the hope to gain new insights into breast cancer biology and thereby improve the performance of current prognostic tools. However, it also poses several serious challenges to classical data analysis techniques related to the characteristics of resulting data, mainly high dimensionality and low signal-to-noise ratio. Despite the tremendous research work performed to handle the first challenge in the feature selection framework, very little attention has been directed to address the second one. We propose in this article to address both issues simultaneously based on symbolic data analysis capabilities in order to derive more accurate genetic marker-based prognostic models. In particular, interval data representation is employed to model various uncertainties in microarray measurements. A recent feature selection algorithm that handles symbolic interval data is used then to derive a genetic signature. The predictive value of the derived signature is then assessed by following a rigorous experimental setup and compared with existing prognostic approaches in terms of predictive performance and estimated survival probability. It is shown that the derived signature (GenSym) performs significantly better than other prognostic models, including the 70-gene signature, St. Gallen, and National Institutes of Health criteria.

  6. A discrete wavelet based feature extraction and hybrid classification technique for microarray data analysis.

    PubMed

    Bennet, Jaison; Ganaprakasam, Chilambuchelvan Arul; Arputharaj, Kannan

    2014-01-01

    Cancer classification by doctors and radiologists was based on morphological and clinical features and had limited diagnostic ability in olden days. The recent arrival of DNA microarray technology has led to the concurrent monitoring of thousands of gene expressions in a single chip which stimulates the progress in cancer classification. In this paper, we have proposed a hybrid approach for microarray data classification based on nearest neighbor (KNN), naive Bayes, and support vector machine (SVM). Feature selection prior to classification plays a vital role and a feature selection technique which combines discrete wavelet transform (DWT) and moving window technique (MWT) is used. The performance of the proposed method is compared with the conventional classifiers like support vector machine, nearest neighbor, and naive Bayes. Experiments have been conducted on both real and benchmark datasets and the results indicate that the ensemble approach produces higher classification accuracy than conventional classifiers. This paper serves as an automated system for the classification of cancer and can be applied by doctors in real cases which serve as a boon to the medical community. This work further reduces the misclassification of cancers which is highly not allowed in cancer detection.

  7. Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

    NASA Technical Reports Server (NTRS)

    Wilson, James W.; Ramamurthy, Rajee; Porwollik, Steffen; McClelland, Michael; Hammond, Timothy; Allen, Pat; Ott, C. Mark; Pierson, Duane L.; Nickerson, Cheryl A.

    2002-01-01

    The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 x g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies.

  8. Metatranscriptomic analysis of the response of river biofilms to pharmaceutical products, using anonymous DNA microarrays.

    PubMed

    Yergeau, Etienne; Lawrence, John R; Waiser, Marley J; Korber, Darren R; Greer, Charles W

    2010-08-01

    Pharmaceutical products are released at low concentrations into aquatic environments following domestic wastewater treatment. Such low concentrations have been shown to induce transcriptional responses in microorganisms, which could have consequences on aquatic ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two rivers of differing trophic statuses and subsequently treated with environmentally relevant doses (ng/liter to microg/liter range) of four pharmaceuticals (erythromycin [ER], gemfibrozil [GM], sulfamethazine [SN], and sulfamethoxazole [SL]). To monitor functional gene expression, we constructed a 9,600-feature anonymous DNA microarray platform onto which cDNA from the biofilms was hybridized. Pharmaceutical treatments induced both positive and negative transcriptional responses from biofilm microorganisms. For instance, ER induced the transcription of several stress, transcription, and replication genes, while GM, a lipid regulator, induced transcriptional responses from several genes involved in lipid metabolism. SN caused shifts in genes involved in energy production and conversion, and SL induced responses from a range of cell membrane and outer envelope genes, which in turn could affect biofilm formation. The results presented here demonstrate for the first time that low concentrations of small molecules can induce transcriptional changes in a complex microbial community. The relevance of these results also demonstrates the usefulness of anonymous DNA microarrays for large-scale metatranscriptomic studies of communities from differing aquatic ecosystems.

  9. Clinical and microarray analysis of breast cancers of all subtypes from two prospective preoperative chemotherapy studies

    PubMed Central

    Okuma, H S; Koizumi, F; Hirakawa, A; Nakatochi, M; Komori, O; Hashimoto, J; Kodaira, M; Yunokawa, M; Yamamoto, H; Yonemori, K; Shimizu, C; Fujiwara, Y; Tamura, K

    2016-01-01

    Background: We aimed to analyse clinical and gene expression profiles to predict pathologic complete response and disease-free survival using two consecutive, prospective, preoperative chemotherapy trial cohorts. Methods: Clinicopathological and gene expression data were evaluated in a cohort from two consecutive phase II preoperative studies that included patients with stage IIA–IIIC breast cancer of all subtypes. Analysed specimens were obtained before preoperative chemotherapy, and cDNA microarray analyses were performed using the Affymetrix Gene Chip U133 plus 2.0. Results: Between December 2005 and December 2010, 122 patients were analysed. The pathologic complete response rate was significantly higher in HER2+ and HR−/HER2− cancers. Age, pathologic complete response, HR−/HER2− status, and lymph node positivity (⩾4) were significant poor prognostic factors for disease-free survival. For the cDNA microarray analyses, sufficient tumour samples were available from 78 of the 107 patients (73%). An 8-gene signature predictive of pathologic complete response and a 17-gene signature predictive of prognosis were identified. Patients were categorised into low-risk (n=45) and high-risk groups (n=33) (HR 70.0, P=0.004). Conclusions: This study yielded preliminary data on the expression of specific genes predicting pathologic complete response and disease-free survival in a cohort of chemonaïve breast cancer patients. Further validation may distinguish those who would benefit most from perioperative chemotherapy as well as those needing further intervention. PMID:27415010

  10. Analysis of microarray-identified genes and microRNAs associated with drug resistance in ovarian cancer.

    PubMed

    Zou, Jing; Yin, Fuqiang; Wang, Qi; Zhang, Wei; Li, Li

    2015-01-01

    The aim of this study was to identify potential microRNAs and genes associated with drug resistance in ovarian cancer through web-available microarrays. The drug resistant-related microRNA microarray dataset GS54665 and mRNA dataset GSE33482, GSE28646, and GSE15372 were downloaded from the Gene Expression Omnibus database. Dysregulated microRNAs/genes were screened with GEO2R and were further identified in SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells by real-time quantitative PCR (qRT-PCR), and then their associations with drug resistance was analyzed by comprehensive bioinformatic analyses. Nine microRNAs (microRNA-199a-5p, microRNA-199a-3p, microRNA-199b-3p, microRNA-215, microRNA-335, microRNA-18b, microRNA-363, microRNA-645 and microRNA-141) and 38 genes were identified to be differentially expressed in drug-resistant ovarian cancer cells, with seven genes (NHSL1, EPHA3, USP51, ZSCAN4, EPHA7, SNCA and PI15) exhibited exactly the same expression trends in all three microarrays. Biological process annotation and pathway enrichment analysis of the 9 microRNAs and 38 genes identified several drug resistant-related signaling pathways, and the microRNA-mRNA interaction revealed the existence of a targeted regulatory relationship between the 9 microRNAs and most of the 38 genes. The expression of 9 microRNAs and the 7 genes by qRT-PCR in SKOV3/DDP and A2780/DDP cells indicating a consistent expression profile with the microarrays. Among those, the expression of EPHA7 and PI15 were negatively correlated with that of microRNA-141, and they were also identified as potential targets of this microRNA via microRNA-mRNA interaction. We thus concluded that microRNA-141, EPHA7, and PI15 might jointly participate in the regulation of drug resistance in ovarian cancer and serve as potential targets in targeted therapies.

  11. Genome-wide microarray analysis of human fibroblasts in response to γ radiation and the radiation-induced bystander effect.

    PubMed

    Kalanxhi, Erta; Dahle, Jostein

    2012-01-01

    Radiation-induced bystander effects have been studied extensively due to their potential implications for cancer therapy and radiation protection; however, a complete understanding of the molecular mechanisms remains to be elucidated. In this study, we monitored transcriptional responses to γ radiation in irradiated and bystander fibroblasts simultaneously employing a genome-wide microarray approach to determine factors that may be modulated in the generation or propagation of the bystander effect. For the microarray data we employed analysis at both the single-gene and gene-set level to place the findings in a biological context. Unirradiated bystander fibroblasts that were recipients of growth medium harvested from irradiated cultures 2 h after exposure to 2 Gy displayed transient enrichment in gene sets belonging to ribosome, oxidative phosphorylation and neurodegenerative disease pathways associated with mitochondrial dysfunctions. The response to direct irradiation was characterized by induction of signaling and apoptosis genes and the gradual formation of a cellular immune response. A set of 14 genes, many of which were regulated by p53, were found to be induced early after irradiation (prior to medium transfer) and may be important in the generation or propagation of the bystander effect.

  12. The mechanisms underlying α-amanitin resistance in Drosophila melanogaster: a microarray analysis.

    PubMed

    Mitchell, Chelsea L; Saul, Michael C; Lei, Liang; Wei, Hairong; Werner, Thomas

    2014-01-01

    The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.

  13. The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

    PubMed Central

    Mitchell, Chelsea L.; Saul, Michael C.; Lei, Liang; Wei, Hairong; Werner, Thomas

    2014-01-01

    The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors. PMID:24695618

  14. Iterative Bayesian Model Averaging: a method for the application of survival analysis to high-dimensional microarray data

    PubMed Central

    Annest, Amalia; Bumgarner, Roger E; Raftery, Adrian E; Yeung, Ka Yee

    2009-01-01

    Background Microarray technology is increasingly used to identify potential biomarkers for cancer prognostics and diagnostics. Previously, we have developed the iterative Bayesian Model Averaging (BMA) algorithm for use in classification. Here, we extend the iterative BMA algorithm for application to survival analysis on high-dimensional microarray data. The main goal in applying survival analysis to microarray data is to determine a highly predictive model of patients' time to event (such as death, relapse, or metastasis) using a small number of selected genes. Our multivariate procedure combines the effectiveness of multiple contending models by calculating the weighted average of their posterior probability distributions. Our results demonstrate that our iterative BMA algorithm for survival analysis achieves high prediction accuracy while consistently selecting a small and cost-effective number of predictor genes. Results We applied the iterative BMA algorithm to two cancer datasets: breast cancer and diffuse large B-cell lymphoma (DLBCL) data. On the breast cancer data, the algorithm selected a total of 15 predictor genes across 84 contending models from the training data. The maximum likelihood estimates of the selected genes and the posterior probabilities of the selected models from the training data were used to divide patients in the test (or validation) dataset into high- and low-risk categories. Using the genes and models determined from the training data, we assigned patients from the test data into highly distinct risk groups (as indicated by a p-value of 7.26e-05 from the log-rank test). Moreover, we achieved comparable results using only the 5 top selected genes with 100% posterior probabilities. On the DLBCL data, our iterative BMA procedure selected a total of 25 genes across 3 contending models from the training data. Once again, we assigned the patients in the validation set to significantly distinct risk groups (p-value = 0.00139). Conclusion

  15. Microarray analysis of the inflammatory and immune responses in head kidney turbot leucocytes treated with resveratrol.

    PubMed

    Domínguez, Berta; Pardo, Belén G; Noia, Manuel; Millán, Adrián; Gómez-Tato, Antonio; Martínez, Paulino; Leiro, José; Lamas, Jesús

    2013-03-01

    A DNA oligo-microarray enriched in genes and involved in inflammatory and immune responses was used to evaluate the effects of resveratrol on gene expression in turbot head kidney leucocytes. Leucocytes were cultured for 3, 6 and 24 h, in the presence or absence of resveratrol, or were stimulated with the membrane fraction of the parasite Philasterides dicentrarchi or with the membrane plus resveratrol. Gene expression changed considerably in control cells, and several of the regulated genes were related to inflammatory and immune responses and to the cytoskeleton. Similar changes in gene expression occurred in control cells and in cells stimulated with P. dicentrarchi membrane fraction. Treatment with resveratrol induced changes in the expression (mostly down-regulation) of several genes involved in immune responses and inflammation. Thus, the down-regulation of the transcription factor PU.1, pentraxin-multidomain protein, heme oxygenase 1, S100 calcium-binding protein A-16 (S100A16) and the signal transducer and activator of transcription 4 was observed after all three incubation times. The down-regulation of the suppressor of cytokine signalling 3a, LPS-induced tumour necrosis alpha, hepcidin, metallothionein, TLR8 and the calcium dependent lectin A was observed after 3 and 6 h. Resveratrol also decreased the expression of CCL20, IL-8, apolipoprotein E and glutathione S-transferase after incubation for 6 and 24 h, and of TNF-α after incubation for 3 and 24 h. Resveratrol also induced strong regulation of several cytoskeleton-related genes. The use of the turbot oligo-microarray enabled us to discover genes whose expression was not previously suspected of being modulated by this polyphenol.

  16. Systematic gene microarray analysis of the lncRNA expression profiles in human uterine cervix carcinoma.

    PubMed

    Chen, Jie; Fu, Ziyi; Ji, Chenbo; Gu, Pingqing; Xu, Pengfei; Yu, Ningzhu; Kan, Yansheng; Wu, Xiaowei; Shen, Rong; Shen, Yan

    2015-05-01

    The human uterine cervix carcinoma is one of the most well-known malignancy reproductive system cancers, which threatens women health globally. However, the mechanisms of the oncogenesis and development process of cervix carcinoma are not yet fully understood. Long non-coding RNAs (lncRNAs) have been proved to play key roles in various biological processes, especially development of cancer. The function and mechanism of lncRNAs on cervix carcinoma is still rarely reported. We selected 3 cervix cancer and normal cervix tissues separately, then performed lncRNA microarray to detect the differentially expressed lncRNAs. Subsequently, we explored the potential function of these dysregulated lncRNAs through online bioinformatics databases. Finally, quantity real-time PCR was carried out to confirm the expression levels of these dysregulated lncRNAs in cervix cancer and normal tissues. We uncovered the profiles of differentially expressed lncRNAs between normal and cervix carcinoma tissues by using the microarray techniques, and found 1622 upregulated and 3026 downregulated lncRNAs (fold-change>2.0) in cervix carcinoma compared to the normal cervical tissue. Furthermore, we found HOXA11-AS might participate in cervix carcinogenesis by regulating HOXA11, which is involved in regulating biological processes of cervix cancer. This study afforded expression profiles of lncRNAs between cervix carcinoma tissue and normal cervical tissue, which could provide database for further research about the function and mechanism of key-lncRNAs in cervix carcinoma, and might be helpful to explore potential diagnosis factors and therapeutic targets for cervix carcinoma.

  17. Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis

    PubMed Central

    Ahn, Woong Shick; Kim, Ko-Woon; Bae, Su Mi; Yoon, Joo Hee; Lee, Joon Mo; Namkoong, Sung Eun; Kim, Jin Hong; Kim, Chong Kook; Lee, Young Joo; Kim, Yong-Wan

    2003-01-01

    This study utilized both cDNA microarray and two-dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six-paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene-expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two-dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid-binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two-dimensional protein analysis via its cellular process-level approach. Therefore, a valuable prognostic candidate gene with relevance to disease-specific pathogenesis can be found at cellular process levels. PMID:14748746

  18. Analyzing Microarray Data.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2017-03-01

    Because there is no widely used software for analyzing RNA-seq data that has a graphical user interface, this protocol provides an example of analyzing microarray data using Babelomics. This analysis entails performing quantile normalization and then detecting differentially expressed genes associated with the transgenesis of a human oncogene c-Myc in mice. Finally, hierarchical clustering is performed on the differentially expressed genes using the Cluster program, and the results are visualized using TreeView.

  19. Contributions to Statistical Problems Related to Microarray Data

    ERIC Educational Resources Information Center

    Hong, Feng

    2009-01-01

    Microarray is a high throughput technology to measure the gene expression. Analysis of microarray data brings many interesting and challenging problems. This thesis consists three studies related to microarray data. First, we propose a Bayesian model for microarray data and use Bayes Factors to identify differentially expressed genes. Second, we…

  20. High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients*

    PubMed Central

    Hecker, Michael; Fitzner, Brit; Wendt, Matthias; Lorenz, Peter; Flechtner, Kristin; Steinbeck, Felix; Schröder, Ina; Thiesen, Hans-Jürgen; Zettl, Uwe Klaus

    2016-01-01

    Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g. MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (p values <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392–411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS. PMID:26831522

  1. Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

    PubMed Central

    Martínez-Soto, Soledad; Legarra, Sheila; Pata-Merci, Noémie; Guegan, Justine; Danglot, Giselle; Bernheim, Alain; Meléndez, Bárbara; Rey, Juan A.; Castresana, Javier S.

    2014-01-01

    Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells. PMID:25392930

  2. Use of microarray analysis to unveil transcription factor and gene networks contributing to Beta cell dysfunction and apoptosis.

    PubMed

    Eizirik, Decio L; Kutlu, Burak; Rasschaert, Joanne; Darville, Martine; Cardozo, Alessandra K

    2003-11-01

    The beta cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat beta cells exposed to the proapoptotic cytokines, IL-1beta and/or IFN-gamma. The picture emerging from these experiments is that beta cells are not passive bystanders of their own destruction. On the contrary, beta cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed beta cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-kappaB. NF-kappaB blocking prevents cytokine-induced beta cell death, and characterization of NF-kappaB-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of beta cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the beta cell "decision" to undergo or not apoptosis in early T1DM.

  3. Microarray analysis on the lncRNA expression profile in male hepatocelluar carcinoma patients with chronic hepatitis B virus infection

    PubMed Central

    Niu, Jianjun; Lin, Yong; Liu, Pingguo; Yu, Yiwen; Su, Chenghao; Wang, Xiaomin

    2016-01-01

    Long non-coding RNAs are involved with development and progression of cancer, and the advance of microarray technology allows the researchers to investigate the complete expression profile of lncRNA in various kinds of sample. We enrolled 5 male primary HCC cases with chronic HBV infection and the HCC and normal tissues have been obtained during the resection surgery. After total RNA extraction, the lncRNA microarray analysis was conducted to determine the lncRNA and mRNA expression signals. 612 lncRNAs and 1,064 mRNAs were significantly up-regulated in HCC tissue while 656 lncRNAs and 1,532 mRNAs were down-regulated in HCC tissues. Compared with normal tissues, XLOC_007433 (fold change: 12.80) and AC144449.1 (fold change: 27.20) were the most over- and under-expressed lncRNAs in HCC tissues. As for the mRNA, THBS4 (fold change:41.13) and CXCL14 (fold change: 58.03) were the most over- and under-expressed mRNAs in HCC tissues when comparing with their normal counterparts. In total, 4,552 pairs of lncRNA-mRNA were identified and the co-expression network was constructed. Moreover, the gene ontology enrichment analysis showed that the significantly different transcript between HCC and normal tissues were mainly associated with response to wounding, inflammatory response, protein hetrodimerization activity, response to stress which involved with biological process and molecular function. The pathway analysis suggested that the most significant pathways consisted of alcoholism, regulatory RNA pathways and RNA polymerase transcription. Several novel differentially expressed lncRNAs and mRNAs were identified in the present study. PMID:27769059

  4. Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

    PubMed

    Quintela, T; Marcelino, H; Deery, M J; Feret, R; Howard, J; Lilley, K S; Albuquerque, T; Gonçalves, I; Duarte, A C; Santos, C R A

    2016-01-01

    The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

  5. Optical and surface analysis of DNA microarrays to assess printed spot heterogeneity

    NASA Astrophysics Data System (ADS)

    Nagaraja Rao, Archana

    DNA microarrays have been plagued with analytical problems with quantitation, metrics, figures of merit, and reliability and reproducibility issues, hindering their acceptance in clinical and diagnostic settings. The main deficiency in the printed DNA format is the microspot heterogeneity occurring during array fabrication and further amplified during target hybridization. Work described in this dissertation focuses on assessment of DNA microarray spots generated with conventional pin-type contact printing of fluorescently labeled DNA probes, on industry-standard commercial polymer-coated array slides and their hybridization with complementary oligomer DNA target. Printing of probe DNA microspots shares many features of commonly reported droplet evaporation dynamics that lead to different drying patterns and spot morphologies. This study directly identifies and analyzes different DNA probe chemical and spatial microenvironments within spots, analyzed with high-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical imaging, confocal epifluorescence, and probe microscopy force imaging methods. Drying of DNA probe spots shows Marangoni flow effects with high densities of probe DNA-Cy3 located in spot centers and nonhomogeneous DNA distributed radially within printed spots with both TOF-SIMS imaging and epifluorescence microscopy. Target hybridization kinetics and duplex formation were assessed using real-time in situ confocal imaging, and confirmed radial hemispherical diffusion-mediated distribution of target capture from spot edge to its interior. Kinetic modeling indicates pseudo-first order kinetics due to transport limitations and local density-dependent probe interactions with diffusing target. Fluorescence resonance energy transfer (FRET) and photobleaching results show that the high- density probe overcrowding in spots facilitates a broad range of target binding interactions regardless of dye orientations. Moreover, lateral probe density

  6. Differential expression of endoglin in human melanoma cells expressing the V3 isoform of versican by microarray analysis.

    PubMed

    Miquel-Serra, Laia; Hernandez, Daniel; Docampo, María Jose; Bassols, Anna

    2010-01-01

    Versican is a large chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The large isoforms V0 and V1 promote melanoma cell proliferation. We previously described that overexpression of the short V3 isoform in MeWo human melanoma cells markedly reduced tumor cell growth in vitro and in vivo, but favored the appearance of secondary tumors. This study aimed to elucidate the mechanisms of V3 by identifying differentially expressed genes between parental and V3-expressing MeWo melanoma cells using microarray analysis. V3 expression significantly reduced the expression of endoglin, a transforming growth factor-β superfamily co-receptor. Other differentially expressed genes were VEGF and PPP1R14B. Changes in endoglin levels were validated by qRT-PCR and Western blotting.

  7. Analysis of differentially expressed genes in placental tissues of preeclampsia patients using microarray combined with the Connectivity Map database.

    PubMed

    Song, Y; Liu, J; Huang, S; Zhang, L

    2013-12-01

    Preeclampsia (PE), which affects 2-7% of human pregnancies, causes significant maternal and neonatal morbidity and mortality. To better understand the pathophysiology of PE, the gene expression profiles of placental tissue from 5 controls and 5 PE patients were assessed using microarray. A total of 224 transcripts were significantly differentially expressed (>2-fold change and q value <0.05, SAM software). Gene Ontology (GO) enrichment analysis indicated that genes involved in hypoxia and oxidative and reductive processes were significantly changed. Three differentially expressed genes (DEGs) involved in these biological processes were further verified by quantitative real-time PCR. Finally, the potential therapeutic agents for PE were explored via the Connectivity Map database. In conclusion, the data obtained in this study might provide clues to better understand the pathophysiology of PE and to identify potential therapeutic agents for PE patients.

  8. Bannayan-Riley-Ruvalcaba Syndrome in a Patient with a PTEN Mutation Identified by Chromosomal Microarray Analysis: A Case Report

    PubMed Central

    Lee, Sun Hwa; Tchah, Hann

    2017-01-01

    Bannayan-Riley-Ruvalcaba syndrome (BRRS) is one of the phosphatase and tensin homolog hamartoma tumor syndrome with a PTEN gene mutation. It is a rare dominant autosomal disorder characterized by cutaneous lipomas, macrocephaly, intestinal polyps, and developmental delay. Diagnosing this syndrome is important, because it may represent the pediatric phenotype of Cowden syndrome, in which there is an increased risk for malignant tumors in children. Until now, the prevalence of BRRS is unknown. Several dozen cases have been reported in the medical literature, but no case has been reported in Korea. Here we report a case of a 19-year-old girl who was diagnosed with BRRS because of macrocephaly, intellectual disability, and intestinal polyps. Her mother had similar findings and a PTEN mutation. Neither patient had mutations detected by conventional mutation-detection techniques, but a PTEN gene deletion was demonstrated by chromosomal microarray analysis.

  9. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces.

  10. Selection of Ceratitis capitata (Diptera: Tephritidae) Specific Recombinant Monoclonal Phage Display Antibodies for Prey Detection Analysis

    PubMed Central

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators. PMID:23272105

  11. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    PubMed

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  12. Effect of Lactobacillus brevis KB290 on the cell-mediated cytotoxic activity of mouse splenocytes: a DNA microarray analysis.

    PubMed

    Fukui, Yuichiro; Sasaki, Erika; Fuke, Nobuo; Nakai, Yuji; Ishijima, Tomoko; Abe, Keiko; Yajima, Nobuhiro

    2013-11-14

    Lactic acid bacteria confer a variety of health benefits. Here, we investigate the mechanisms by which Lactobacillus brevis KB290 (KB290) enhances cell-mediated cytotoxic activity. Female BALB/c mice aged 9 weeks were fed a diet containing KB290 (3 × 10(9) colony-forming units/g) or starch for 1 d. The resulting cytotoxic activity of splenocytes against YAC-1 cells was measured using flow cytometry and analysed for gene expression using DNA microarray technology. KB290 enhanced the cell-mediated cytotoxic activity of splenocytes. DNA microarray analysis identified 327 up-regulated and 347 down-regulated genes that characterised the KB290 diet group. The up-regulated genes were significantly enriched in Gene Ontology terms related to immunity, and, especially, a positive regulation of T-cell-mediated cytotoxicity existed among these terms. Almost all the genes included in the term encoded major histocompatibility complex (MHC) class I molecules involved in the presentation of antigen to CD8(+) cytotoxic T cells. Marco and Signr1 specific to marginal zone macrophages (MZM), antigen-presenting cells, were also up-regulated. Flow cytometric analysis confirmed that the proportion of MZM was significantly increased by KB290 ingestion. Additionally, the over-represented Kyoto Encyclopedia of Genes and Genomes pathways among the up-regulated genes were those for natural killer (NK) cell-mediated cytotoxicity and antigen processing and presentation. The results for the selected genes associated with NK cells and CD8(+) cytotoxic T cells were confirmed by quantitative RT-PCR. These results suggest that enhanced cytotoxic activity could be caused by the activation of NK cells and/or of CD8(+) cytotoxic T cells stimulated via MHC class I presentation.

  13. Lectin-based protein microarray analysis of differences in serum alpha-2-macroglobulin glycosylation between patients with colorectal cancer and persons without cancer.

    PubMed

    Šunderić, Miloš; Šedivá, Alena; Robajac, Dragana; Miljuš, Goran; Gemeiner, Peter; Nedić, Olgica; Katrlík, Jaroslav

    2016-07-01

    Glycosylation is co- and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin-based protein microarray method enabling determination of differences in protein glycosylation. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin-based protein microarray was applied in investigation of differences in alpha-2-macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Student's t-test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N-acetylglucosamine and mannose residues, and tri-/tetraantennary complex type high-mannose N-glycans. A novel lectin-based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high-throughput determination of differences in protein glycosylation isolated from serum or other samples.

  14. Fucosyltransferase 8 expression in breast cancer patients: A high throughput tissue microarray analysis.

    PubMed

    Yue, Liling; Han, Cuicui; Li, Zubin; Li, Xin; Liu, Deshui; Liu, Shulin; Yu, Haitao

    2016-05-01

    The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (p<0.001). The percent of FUT8 staining in breast cancer tissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients.

  15. Benefits of in-situ synthesized microarrays for analysis of gene expression in understudied microorganisms.

    PubMed

    Postier, Bradley; Didonato, Raymond; Nevin, Kelly P; Liu, Anna; Frank, Bryan; Lovley, Derek; Methe, Barbara A

    2008-07-01

    Although the genome sequences of many microorganisms are now known, whole-genome DNA microarray platforms consisting of PCR amplicon, or oligonucleotide elements printed onto glass slides have been readily available for only a relatively few, highly studied microorganisms. For those microorganisms more recently cultured or studied by fewer investigators it has been difficult to justify the initial time and expense of developing such array platforms especially if only a limited number of gene expression studies are envisioned. However, in-situ synthesized oligonucleotide (ISO) arrays can be inexpensively fabricated on an 'as needed' basis with a reduced initial investment in time, personnel, resources, and costs. To evaluate the performance of one ISO array platform, gene expression patterns in Geobacter sulfurreducens under nitrogen-fixing conditions were compared with results from quantitative reverse transcriptase PCR (qRT-PCR) and previously published data from a similar experiment using spotted PCR amplicon arrays. There were strong correlations between the results of the ISO arrays and the results from qRT-PCR (r(2)=0.762) and spotted array (r(2)=0.744) analyses. After initial use the ISO arrays could be successfully stripped and reused. The increased flexibility in array design and reusability coupled with a lower initial investment in terms of fabrication time and cost for the ISO arrays suggest that they may be the preferred approach when investigating gene expression in microorganisms, especially when only a few expression studies are required.

  16. Global Microarray Analysis of Alkaliphilic Halotolerant Bacterium Bacillus sp. N16-5 Salt Stress Adaptation.

    PubMed

    Yin, Liang; Xue, Yanfen; Ma, Yanhe

    2015-01-01

    The alkaliphilic halotolerant bacterium Bacillus sp. N16-5 is often exposed to salt stress in its natural habitats. In this study, we used one-colour microarrays to investigate adaptive responses of Bacillus sp. N16-5 transcriptome to long-term growth at different salinity levels (0%, 2%, 8%, and 15% NaCl) and to a sudden salt increase from 0% to 8% NaCl. The common strategies used by bacteria to survive and grow at high salt conditions, such as K+ uptake, Na+ efflux, and the accumulation of organic compatible solutes (glycine betaine and ectoine), were observed in Bacillus sp. N16-5. The genes of SigB regulon involved in general stress responses and chaperone-encoding genes were also induced by high salt concentration. Moreover, the genes regulating swarming ability and the composition of the cytoplasmic membrane and cell wall were also differentially expressed. The genes involved in iron uptake were down-regulated, whereas the iron homeostasis regulator Fur was up-regulated, suggesting that Fur may play a role in the salt adaption of Bacillus sp. N16-5. In summary, we present a comprehensive gene expression profiling of alkaliphilic Bacillus sp. N16-5 cells exposed to high salt stress, which would help elucidate the mechanisms underlying alkaliphilic Bacillus spp. survival in and adaptation to salt stress.

  17. Probabilistic non-negative matrix factorization: theory and application to microarray data analysis.

    PubMed

    Bayar, Belhassen; Bouaynaya, Nidhal; Shterenberg, Roman

    2014-02-01

    Non-negative matrix factorization (NMF) has proven to be a useful decomposition technique for multivariate data, where the non-negativity constraint is necessary to have a meaningful physical interpretation. NMF reduces the dimensionality of non-negative data by decomposing it into two smaller non-negative factors with physical interpretation for class discovery. The NMF algorithm, however, assumes a deterministic framework. In particular, the effect of the data noise on the stability of the factorization and the convergence of the algorithm are unknown. Collected data, on the other hand, is stochastic in nature due to measurement noise and sometimes inherent variability in the physical process. This paper presents new theoretical and applied developments to the problem of non-negative matrix factorization (NMF). First, we generalize the deterministic NMF algorithm to include a general class of update rules that converges towards an optimal non-negative factorization. Second, we extend the NMF framework to the probabilistic case (PNMF). We show that the Maximum a posteriori (MAP) estimate of the non-negative factors is the solution to a weighted regularized non-negative matrix factorization problem. We subsequently derive update rules that converge towards an optimal solution. Third, we apply the PNMF to cluster and classify DNA microarrays data. The proposed PNMF is shown to outperform the deterministic NMF and the sparse NMF algorithms in clustering stability and classification accuracy.

  18. High-density tiling microarray analysis of the full transcriptional activity of yeast.

    PubMed

    David, Lior; Clauder-Münster, Sandra; Steinmetz, Lars M

    2014-01-01

    Understanding the relationship between DNA sequence variation and phenotypic variation in complex or quantitative traits is one of the major challenges in modern biology. We are witnessing a deluge of DNA sequence information and association studies of genetic polymorphisms with phenotypes of interest in families and populations. In addition, it has become clear that large portions of eukaryotic genomes beyond protein-coding genes are transcribed, generating numerous noncoding RNA (ncRNA) molecules whose functions remain mostly unknown.DNA oligonucleotide microarrays constitute a powerful technology for studying the expression of genes in different organisms. The Saccharomyces cerevisiae tiling array presents a significant advance over previous array-based platforms. It has a high density of overlapping probes that start on average every 8 bp along each strand of the genome, enabling precise definition of transcript structure. Furthermore, the array includes probes specific for the polymorphic positions of another, distantly related yeast strain, allowing accurate measurement of allele-specific expression in a hybrid of the two strains. This technology thus allows high-resolution, quantitative, strand- and allele-specific measurements of transcription from a full eukaryotic genome. In this chapter, we describe the methods for extracting RNA, synthesizing first-strand cDNA, fragmenting, and labeling of samples for hybridization to the tiling array. Combining genome-wide information on variation in DNA sequence with variation in transcript structure and levels promises to increase our understanding of the genotype-to-phenotype relationship.

  19. Microarray Analysis of Gene Expression at the Tumor Front of Colon Cancer.

    PubMed

    Kobayashi, Takaaki; Masaki, Tadahiko; Nozaki, Eriko; Sugiyama, Masanori; Nagashima, Fumio; Furuse, Junji; Onishi, Hiroaki; Watanabe, Takashi; Ohkura, Yasuo

    2015-12-01

    Budding or the presence poorly differentiated clusters at the boundary of cancer tissue is a pathologically important finding and serves as a prognostic factor in colorectal cancer. However, few studies have examined the cancer tissue boundary in clinical samples. The purpose of the present study was to examine gene expression at the tumor front of colon cancer in surgically resected samples. Cancer tissues were obtained by laser microdissection of 20 surgically resected specimens. Genes with significantly different microarray signals between the tumor front and the tumor center were identified. Among genes showing significant up-regulation at the tumor front were six chemokines [chemokine c-c motif ligand (CCL)2 and -18, chemokine (C-X-C motif) ligand (CXCL)9-11, and interleukin 8 (IL8)], and two apoptosis-related molecules [ubiquitin D (UBD) and baculoviral iap repeat-containing 3 (BIRC3)]. Expression of laminin gamma 2 (LAMC2), matrix metallopeptidase 7 (MMP7) and epithelial-mesenchymal transition (EMT)-related molecules were elevated in the tumor front, but their fold changes were smaller than those of the aforementioned genes. These results suggest that chemokines, in addition to EMT-related molecules, may play important roles in invasion of colon cancer.

  20. Spatio-Temporal Analysis of Cell-Cell Signaling in a Living Cell Microarray

    NASA Astrophysics Data System (ADS)

    Mirsaidov, Utkur; Timp, Winston; Timp, Kaethe; Matsudaira, Paul; Timp, Greg

    2007-03-01

    Cell-cell signaling plays a central role in biology, enabling individual cells to coordinate their activities. For example, bacteria show evidence of intercellular signaling through quorum sensing, a regulatory mechanism that launches a coordinated response, depending on the population density. To explore the spatio-temporal development of cell-to-cell signaling, we have created regular, heterotypic microarrays of living cells in hydrogel using time-multiplexed optical traps for submicron positional control of the cell orientation and location without loss of viability. We studied the Lux system for quorum sensing; splitting it into sender and receiver plasmids, which were subsequently introduced into E. Coli. Induced by IPTG, the sender cells express a fluorescent reporter (mRFP1) and the LuxI enzyme that catalyzes the synthesis of a molecular signal AHL that diffuses through the cell membrane and the extra-cellular scaffold. The receiver cells collect the AHL signal that binds to the LuxR regulator and reports it through GFP production. We have measured the time-delay between the onset of mRFP1 and GFP dependence on intercellular spacing in the array.

  1. Exploring host-pathogen interactions through genome wide protein microarray analysis

    PubMed Central

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F.; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J.; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-01-01

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis. PMID:27302108

  2. Exploring host-pathogen interactions through genome wide protein microarray analysis.

    PubMed

    Scietti, Luigi; Sampieri, Katia; Pinzuti, Irene; Bartolini, Erika; Benucci, Barbara; Liguori, Alessia; Haag, Andreas F; Lo Surdo, Paola; Pansegrau, Werner; Nardi-Dei, Vincenzo; Santini, Laura; Arora, Seguinde; Leber, Xavier; Rindi, Simonetta; Savino, Silvana; Costantino, Paolo; Maione, Domenico; Merola, Marcello; Speziale, Pietro; Bottomley, Matthew J; Bagnoli, Fabio; Masignani, Vega; Pizza, Mariagrazia; Scharenberg, Meike; Schlaeppi, Jean-Marc; Nissum, Mikkel; Liberatori, Sabrina

    2016-06-15

    During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.

  3. Identification of genes associated with tumorigenesis of meibomian cell carcinoma by microarray analysis.

    PubMed

    Kumar, Arun; Kumar Dorairaj, Syril; Prabhakaran, Venkatesh C; Prakash, D Ravi; Chakraborty, Sanjukta

    2007-11-01

    Meibomian cell carcinoma (MCC) is a malignant tumor of the meibomian glands located in the eyelids. No information exists on the cytogenetic and genetic aspects of MCC. There is no report on the gene expression profile of MCC. Thus there is a need, for both scientific and clinical reasons, to identify genes and pathways that are involved in the development and progression of MCC. We analyzed the gene expression profile of MCC by the microarray technique. Forty-four genes were upregulated and 149 genes were downregulated in MCC. Differential expression data were confirmed for 5 genes by semiquantitative RT-PCR in MCC tumors: GTF2H4, RBM12, UBE2D3, DDX17, and LZTS1. We found dysregulation of two major pathways in MCC: MAPK and JAK/STAT. Clusters of genes on chromosomes 1, 12, and 19 were dysregulated in MCC. The data presented here will facilitate the identification of specific markers and therapeutic targets for the treatment of MCC patients.

  4. Microarray based analysis of temperature and oxidative stress induced messenger RNA in Schistosoma mansoni

    PubMed Central

    Aragon, Anthony D.; Imani, Reza A.; Blackburn, Vint R.; Cunningham, Charles

    2008-01-01

    The body’s defense against schistosome infection can take many forms. For example, upon developing acute schistosomiasis, patients often have fever coinciding with larval maturation, migration and early oviposition. As the infection becomes established, the parasite comes under oxidative stress generated by the host immune system. The most common treatment for schistosomiasis is the anti-helminthic drug praziquantel. Its effectiveness, however, is limited due to its inability to kill schistosomes 2 – 4 weeks post-infection. Clearly there is a need for new antischistosomal drugs. We hypothesize that gene products expressed as part of a protective response against heat and/or oxidative stress are potential therapeutic targets for future drug development. Using a 12,166 element oligonucleotide microarray to characterize Schistosoma mansoni genes induced by heat and oxidative stress we found that 1,878 S. mansoni elements were significantly induced by heat stress. These included previously reported heat-shock genes expressing homologs of HSP40, HSP70 and HSP86. One thousand and one elements were induced by oxidative stress including those expressing homologs of superoxide dismutase, glutathione peroxidase and aldehyde dehydrogenase. Seventy-two elements were common to both stressors and could potentially be exploited in the development of novel anti-schistosomal therapeutics. PMID:18775750

  5. Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization

    PubMed Central

    2010-01-01

    Background Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. Results The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of ≥ 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. Conclusions In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae. PMID:20233401

  6. Stronger inflammatory/cytotoxic T cell response in women identified by microarray analysis

    PubMed Central

    Hewagama, Anura; Patel, Dipak; Yarlagadda, Sushma; Strickland, Faith M.; Richardson, Bruce C.

    2009-01-01

    Women develop chronic inflammatory autoimmune diseases more often than men. The mechanisms causing the increased susceptibility are incompletely understood. Chronic immune stimulation characterizes many autoimmune disorders. We hypothesized that repeated stimulation may cause a different T cell response in women than men. Microarrays were used to compare gene expression in T cells from healthy men and women with and without repeated stimulation. Four days following a single stimulation only 25% of differentially expressed, gender-biased genes were expressed at higher levels in women. In contrast, following restimulation 72% were more highly expressed in women. Immune response genes were significantly over-represented among the genes upregulated in women and among the immune response genes, the inflammatory/cytotoxic effector genes interferon gamma (IFNG), lymphotoxin beta (LTB), granzyme A (GZMA), interleukin-12 receptor beta2 (IL12RB2), and granulysin (GNLY) were among those overexpressed to the greatest degree. In contrast, IL17A was the only effector gene more highly expressed in men. Estrogen response elements were identified in the promoters of half the overexpressed immune genes in women, and in <10% of the male biased genes. The differential expression of inflammatory/cytotoxic effector molecules in restimulated female T cells may contribute to the differences in autoimmune diseases between women and men. PMID:19279650

  7. Stronger inflammatory/cytotoxic T-cell response in women identified by microarray analysis.

    PubMed

    Hewagama, A; Patel, D; Yarlagadda, S; Strickland, F M; Richardson, B C

    2009-07-01

    Women develop chronic inflammatory autoimmune diseases more often than men. The mechanisms causing the increased susceptibility are incompletely understood. Chronic immune stimulation characterizes many autoimmune disorders. We hypothesized that repeated stimulation may cause a different T-cell response in women than in men. Microarrays were used to compare gene expression in T cells from healthy men and women with and without repeated stimulation. Four days after a single stimulation, only 25% of differentially expressed, gender-biased genes were expressed at higher levels in women. In contrast, after restimulation, 72% were more highly expressed in women. Immune response genes were significantly over-represented among the genes upregulated in women and among the immune response genes, the inflammatory/cytotoxic effector genes interferon-gamma (IFN-gamma), lymphotoxin beta (LTbeta), granzyme A (GZMA), interleukin-12 receptor beta2 (IL12Rbeta2), and granulysin (GNLY) were among those overexpressed to the highest degree. In contrast, IL17A was the only effector gene more highly expressed in men. Estrogen response elements were identified in the promoters of half the overexpressed immune genes in women, and in <10% of the male-biased genes. The differential expression of inflammatory/cytotoxic effector molecules in restimulated female T cells may contribute to the differences in autoimmune diseases between women and men.

  8. Self-Directed Student Research through Analysis of Microarray Datasets: A Computer-Based Functional Genomics Practical Class for Masters-Level Students

    ERIC Educational Resources Information Center

    Grenville-Briggs, Laura J.; Stansfield, Ian

    2011-01-01

    This report describes a linked series of Masters-level computer practical workshops. They comprise an advanced functional genomics investigation, based upon analysis of a microarray dataset probing yeast DNA damage responses. The workshops require the students to analyse highly complex transcriptomics datasets, and were designed to stimulate…

  9. Microarray analysis of active cardiac remodeling genes in a familial hypertrophic cardiomyopathy mouse model rescued by a phospholamban knockout

    PubMed Central

    Rajan, Sudarsan; Pena, James R.; Jegga, Anil G.; Aronow, Bruce J.; Wolska, Beata M.

    2013-01-01

    Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function. Our laboratories have previously developed two mouse models that affect cardiac performance. One mouse model encodes an FHC-associated mutation in α-tropomyosin: Glu → Gly at amino acid 180, designated as Tm180. These mice display a phenotype that is characteristic of FHC, including severe cardiac hypertrophy with fibrosis and impaired physiological performance. The other model was a gene knockout of phospholamban (PLN KO), a regulator of calcium uptake in the sarcoplasmic reticulum of cardiomyocytes; these hearts exhibit hypercontractility with no pathological abnormalities. Previous work in our laboratories shows that when mice were genetically crossed between the PLN KO and Tm180, the progeny (PLN KO/Tm180) display a rescued hypertrophic phenotype with improved morphology and cardiac function. To understand the changes in gene expression that occur in these models undergoing cardiac remodeling (Tm180, PLN KO, PLN KO/Tm180, and nontransgenic control mice), we conducted microarray analyses of left ventricular tissue at 4 and 12 mo of age. Expression profiling reveals that 1,187 genes changed expression in direct response to the three genetic models. With these 1,187 genes, 11 clusters emerged showing normalization of transcript expression in the PLN KO/Tm180 hearts. In addition, 62 transcripts are highly involved in suppression of the hypertrophic phenotype. Confirmation of the microarray analysis was conducted by quantitative RT-PCR. These results provide insight into genes that alter expression during cardiac remodeling and are active during modulation of the cardiomyopathic phenotype. PMID:23800848

  10. Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

    PubMed Central

    Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

    2012-01-01

    Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

  11. An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis

    PubMed Central

    Wang, Shuaiqin; Sun, Yujia; Liu, Yan; Xiang, Guangxin; Wang, Lei; Cheng, Jing; Liu, Peng

    2015-01-01

    We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis. PMID:25825617

  12. Microarray analysis of tomato plants exposed to the nonviruliferous or viruliferous whitefly vector harboring Pepper golden mosaic virus.

    PubMed

    Musser, Richard O; Hum-Musser, Sue M; Gallucci, Matthew; DesRochers, Brittany; Brown, Judith K

    2014-01-01

    Plants are routinely exposed to biotic and abiotic stresses to which they have evolved by synthesizing constitutive and induced defense compounds. Induced defense compounds are usually made, initially, at low levels; however, following further stimulation by specific kinds of biotic and abiotic stresses, they can be synthesized in relatively large amounts to abate the particular stress. cDNA microarray hybridization was used to identify an array of genes that were differentially expressed in tomato plants 15 d after they were exposed to feeding by nonviruliferous whiteflies or by viruliferous whiteflies carrying Pepper golden mosaic virus (PepGMV) (Begomovirus, Geminiviridae). Tomato plants inoculated by viruliferous whiteflies developed symptoms characteristic of PepGMV, whereas plants exposed to nonviruliferous whitefly feeding or nonwounded (negative) control plants exhibited no disease symptoms. The microarray analysis yielded over 290 spotted probes, with significantly altered expression of 161 putative annotated gene targets, and 129 spotted probes of unknown identities. The majority of the differentially regulated "known" genes were associated with the plants exposed to viruliferous compared with nonviruliferous whitefly feeding. Overall, significant differences in gene expression were represented by major physiological functions including defense-, pathogen-, photosynthesis-, and signaling-related responses and were similar to genes identified for other insect-plant systems. Viruliferous whitefly-stimulated gene expression was validated by real-time quantitative polymerase chain reaction of selected, representative candidate genes (messenger RNA): arginase, dehydrin, pathogenesis-related proteins 1 and -4, polyphenol oxidase, and several protease inhibitors. This is the first comparative profiling of the expression of tomato plants portraying different responses to biotic stress induced by viruliferous whitefly feeding (with resultant virus infection

  13. A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy.

    PubMed

    Qin, Ying; Tian, Ya-Ping

    2010-06-01

    The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.

  14. Resistance exercise training influences skeletal muscle immune activation: a microarray analysis

    PubMed Central

    Liu, Dongmei; Sartor, Maureen A.; IglayReger, Heidi B.; Pistilli, Emidio E.; Gutmann, Laurie; Nader, Gustavo A.; Hoffman, Eric P.

    2012-01-01

    The primary aim of this investigation was to evaluate the effect of training on the immune activation in skeletal muscle in response to an acute bout of resistance exercise (RE). Seven young healthy men and women underwent a 12-wk supervised progressive unilateral arm RE training program. One week after the last training session, subjects performed an acute bout of bilateral RE in which the trained and the untrained arm exercised at the same relative intensity. Muscle biopsies were obtained 4 h postexercise from the biceps brachii of both arms and assessed for global transcriptom using Affymetrix U133 plus 2.0 microarrays. Significantly regulated biological processes and gene groups were analyzed using a logistic regression-based method following differential (trained vs. untrained) gene expression testing via an intensity-based Bayesian moderated t-test. The results from the present study suggest that training blunts the transcriptional upregulation of immune activation by minimizing expression of genes involved in monocyte recruitment and enhancing gene expression involved in macrophage anti-inflammatory polarization. Additionally, our data suggest that training blunts the transcriptional upregulation of the stress response and the downregulation of glucose metabolism, mitochondrial structure, and oxidative phosphorylation, and it enhances the transcriptional upregulation of the extracellular matrix and cytoskeleton development and organization and the downregulation of gene transcription and muscle contraction. This study provides novel insight into the molecular processes involved in the adaptive response of skeletal muscle following RE training and the cellular and molecular events implicating the protective role of training on muscle stress and damage inflicted by acute mechanical loading. PMID:22052873

  15. DNA Microarray Analysis of Anaerobic Methanosarcina Barkeri Reveals Responses to Heat Shock and Air Exposure

    SciTech Connect

    Zhang, Weiwen; Culley, David E.; Nie, Lei; Brockman, Fred J.

    2006-04-08

    Summary Methanosarcina barkeri can grow only under strictly anoxic conditions because enzymes in methane formation pathways of are very oxygen sensitive. However, it has been determined that M. barkeri can survive oxidative stress. To obtain further knowledge of cellular changes in M. barkeri in responsive to oxidative and other environmental stress, a first whole-genome M. barkeri oligonucleotide microarray was constructed according to the draft genome sequence that contains 5072 open reading frames (ORFs) and was used to investigate the global transcriptomic response of M. barkeri to oxidative stress and heat shock. The result showed that 552 genes in the M. barkeri genome were responsive to oxidative stress, while 177 genes responsive to heat-shock, respectively using a cut off of 2.5 fold change. Among them, 101 genes were commonly responsive to both environmental stimuli. In addition to various house-keeping genes, large number of functionally unknown genes (38-57% of total responsive genes) was regulated by both stress conditions. The result showed that the Hsp60 (GroEL) system, which was previously thought not present in archaea, was up-regulated and may play important roles in protein biogenesis in responsive to heat shock in M. barkeri. No gene encoding superoxide dismutase, catalase, nonspecific peroxidases or thioredoxin reductase was differentially expressed when subjected to oxidative stress. Instead, significant downregulation of house-keeping genes and up-regulation of genes encoding transposase was found in responsive to oxidative stress, suggesting that M. barkeri may be adopting a passive protective mechanism by slowing down cellular activities to survive the stress rather than activating a means against oxidative stress.

  16. Effect of leucine uptake on hepatic and skeletal muscle gene expression in rats: a microarray analysis

    PubMed Central

    Cheon, Wookwang

    2015-01-01

    [Purpose] This study was performed to explore the physiological functions of leucine by exploring genes with leucine-dependent variability using DNA microarray. [Methods] Sprague-Dawley rats (n = 20) were separated into a HPD (30% High Protein Diet, n = 10) group and a NPD (0% Non Protein Diet, n = 10) group and fed a protein diet for 2 weeks. At the end of the 2-week period, the rats were fasted for 12-16 hours, further separated into subgroups within the HPD (Saline, n = 5, Leucine, n = 5) and NPD (Saline, n = 5, Leucine, n = 5) groups and administered with a leucine solution. The liver and muscles were harvested after 2 hours for RNA extraction. RNA purification from the isolated muscles and target gene identification using DNA chip were performed. The target gene was determined based on the results of the DNA chip experiment, and mRNA expression of the target gene was analyzed using Real-Time PCR. [Results] In the skeletal muscle, 27 genes were upregulated while 52 genes were down regulated after leucine administration in the NPD group. In the liver, 160 genes were up-regulated while 126 were down-regulated. The per2 gene was one of the genes with leucine-dependent induction in muscles and liver. [Conclusion] This study was performed to explore the physiological functions of leucine, however, a large number of genes showed variability. Therefore, it was difficult to definitively identify the genes linked with a particular physiological function. Various nutritional effects of leucine were observed. High variability in cytokines, receptors, and various membrane proteins were observed, which suggests that leucine functions as more than a nutrient. The interpretation may depend on investigators’ perspectives, therefore, discussion with relevant experts and the BCAA (Branched-Chain Amino Acids) society may be needed for effective utilization of this data. PMID:26244133

  17. In Silico Analysis of Microarray-Based Gene Expression Profiles Predicts Tumor Cell Response to Withanolides

    PubMed Central

    Efferth, Thomas; Greten, Henry Johannes

    2012-01-01

    Withania somnifera (L.) Dunal (Indian ginseng, winter cherry, Solanaceae) is widely used in traditional medicine. Roots are either chewed or used to prepare beverages (aqueous decocts). The major secondary metabolites of Withania somnifera are the withanolides, which are C-28-steroidal lactone triterpenoids. Withania somnifera extracts exert chemopreventive and anticancer activities in vitro and in vivo. The aims of the present in silico study were, firstly, to investigate whether tumor cells develop cross-resistance between standard anticancer drugs and withanolides and, secondly, to elucidate the molecular determinants of sensitivity and resistance of tumor cells towards withanolides. Using IC50 concentrations of eight different withanolides (withaferin A, withaferin A diacetate, 3-azerininylwithaferin A, withafastuosin D diacetate, 4-B-hydroxy-withanolide E, isowithanololide E, withafastuosin E, and withaperuvin) and 19 established anticancer drugs, we analyzed the cross-resistance profile of 60 tumor cell lines. The cell lines revealed cross-resistance between the eight withanolides. Consistent cross-resistance between withanolides and nitrosoureas (carmustin, lomustin, and semimustin) was also observed. Then, we performed transcriptomic microarray-based COMPARE and hierarchical cluster analyses of mRNA expression to identify mRNA expression profiles predicting sensitivity or resistance towards withanolides. Genes from diverse functional groups were significantly associated with response of tumor cells to withaferin A diacetate, e.g. genes functioning in DNA damage and repair, stress response, cell growth regulation, extracellular matrix components, cell adhesion and cell migration, constituents of the ribosome, cytoskeletal organization and regulation, signal transduction, transcription factors, and others. PMID:27605335

  18. Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas

    PubMed Central

    Saghizadeh, Mehrnoosh; Kramerov, Andrei A.; Tajbakhsh, Jian; Aoki, Annette M.; Wang, Charles; Chai, Ning-Ning; Ljubimova, Julia Y.; Sasaki, Takako; Sosne, Gabriel; Carlson, Marc R. J.; Nelson, Stanley F.

    2005-01-01

    PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I. METHODS. RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agi-lent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10. RESULTS. More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin α4 chain, and thymosin β4 genes were down-regulated. The data were corroborated by QPCR and immuno-histochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin α3β1, whereas in normal corneas MMP-10 decreased laminin-10 and integrin α3β1 expression. CONCLUSIONS. Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy. PMID:16186340

  19. Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver

    PubMed Central

    Guillén, Natalia; Navarro, María A.; Arnal, Carmen; Noone, Enda; Arbonés-Mainar, José M.; Acín, Sergio; Surra, Joaquín C.; Muniesa, Pedro; Roche, Helen M.; Osada, Jesús

    2009-01-01

    Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote fatty liver in mice, while cis-9, trans-11-CLA ameliorates this effect, suggesting regulation of multiple genes. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid isomers, and their hepatic gene expression was analyzed with DNA microarrays. To provide an initial screening of candidate genes, only 12 with remarkably modified expression between both CLA isomers were considered and confirmed by quantitative RT-PCR. Additionally mRNA expression of 15 genes involved in lipid metabolism was also studied. Ten genes (Fsp27, Aqp4, Cd36, Ly6d, Scd1, Hsd3b5, Syt1, Cyp7b1, and Tff3) showed significant associations among their expressions and the degree of hepatic steatosis. Their involvement was also analyzed in other models of steatosis. In hyperhomocysteinemic mice lacking Cbs gene, only Fsp27, Cd36, Scd1, Syt1, and Hsd3b5 hepatic expressions were associated with steatosis. In apoE-deficient mice consuming olive-enriched diet displaying reduction of the fatty liver, only Fsp27 and Syt1 expressions were found associated. Using this strategy, we have shown that expression of these genes is highly associated with hepatic steatosis in a genetic disease such as Cbs deficiency and in two common situations such as Western diets containing CLA isomers or a Mediterranean-type diet. Conclusion: The results highlight new processes involved in lipid handling in liver and will help to understand the complex human pathology providing new proteins and new strategies to cope with hepatic steatosis. PMID:19258494

  20. Gene expression microarray analysis of the sciatic nerve of mice with diabetic neuropathy

    PubMed Central

    ZHANG, LEI; QU, SHEN; LIANG, AIBIN; JIANG, HONG; WANG, HAO

    2015-01-01

    The present study aimed to explore novel target genes that regulate the development of diabetic neuropathy (DN) by analyzing gene expression profiles in the sciatic nerve of infected mice. The GSE11343 microarray dataset, which was downloaded from Gene Expression Omnibus, included data on 4 control samples and 5 samples from mice with diabetes induced by streptozotocin (STZ), 5 samples from normal mice treated with rosiglitazone (Rosi) and 5 samples from mice with diabetes induced by STZ and treated with Rosi. Differentially expressed genes (DEGs) between the different groups were identified using the substitution augmentation modification redefinition (SAMR) model. The Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Regulatory and protein-protein interaction networks were searched using BioCarta and STRING, respectively. The protein structures of potential regulatory genes were predicted using the SYBYL program. Compared with the controls, 1,384 DEGs were identified in the mice with STZ-induced diabetes and 7 DEGs were identified in the mice treated with Rosi. There were 518 DEGs identified between the mice in the STZ + Rosi and STZ groups. We identified 45 GO items, and the calmodulin nerve phosphatase and chemokine signaling pathways were identified as the main pathways. Three genes [myristoylated alanine-rich protein kinase C substrate (Marcks), GLI pathogenesis-related 2 (Glipr2) and centrosomal protein 170 kDa (Cep170)] were found to be co-regulated by both STZ and Rosi, the protein structure of which was predicted and certain binding activity to Rosi was docked. Our study demonstrates that the Marcks, Glipr2 and Cep170 genes may be underlying drug targets in the treatment of DN. PMID:25435094

  1. Microarray Analysis of Natural Socially-Regulated Plasticity in Circadian Rhythms of Honey Bees

    PubMed Central

    Rodriguez-Zas, Sandra L.; Southey, Bruce R.; Shemesh, Yair; Rubin, Elad B.; Cohen, Mira; Robinson, Gene E.; Bloch, Guy

    2012-01-01

    Honey bee workers care for ("nurse") the brood around the clock without circadian rhythmicity, but then they forage outside with strong circadian rhythms and a consolidated nightly rest. This chronobiological plasticity is associated with variation in the expression of the canonical “clock genes” that regulate the circadian clock: nurse bees show no brain rhythms of expression, while foragers do. These results suggest that the circadian system is organized differently in nurses and foragers. Nurses switch to activity with circadian rhythms shortly after removed from the hive suggesting that at least some clock cells in their brain continue to measure time while in the hive. We performed a microarray genome-wide survey to determine general patterns of brain gene expression in nurses and foragers sampled around the clock. We found 160 and 541 transcripts that exhibited significant sinusoidal oscillations in nurses and foragers, respectively, with peaks of expression distributed throughout the day in both task groups. Consistent with earlier studies, transcripts of genes involved in circadian rhythms, including Clockwork Orange that has not been studied before in bees, oscillated in foragers but not in nurses. The oscillating transcripts also were enriched for genes involved in the visual system, “development” and “response to stimuli” (foragers), “muscle contraction” and “microfilament motor gene expression” (nurses), and “generation of precursor metabolites” and “energy” (both). Transcripts of genes encoding P450 enzymes oscillated in both nurses and foragers but with a different phase. This study identified new putative clock-controlled genes in the honey bee and suggests that some brain functions show circadian rhythmicity even in nurse bees that are active around the clock. PMID:22306970

  2. Transcriptional Homeostasis of a Mangrove Species, Ceriops tagal, in Saline Environments, as Revealed by Microarray Analysis

    PubMed Central

    Liang, Shan; Fang, Lu; Zhou, Renchao; Tang, Tian; Deng, Shulin; Dong, Suisui; Huang, Yelin; Zhong, Cairong; Shi, Suhua

    2012-01-01

    Background Differential responses to the environmental stresses at the level of transcription play a critical role in adaptation. Mangrove species compose a dominant community in intertidal zones and form dense forests at the sea-land interface, and although the anatomical and physiological features associated with their salt-tolerant lifestyles have been well characterized, little is known about the impact of transcriptional phenotypes on their adaptation to these saline environments. Methodology and Principal findings We report the time-course transcript profiles in the roots of a true mangrove species, Ceriops tagal, as revealed by a series of microarray experiments. The expression of a total of 432 transcripts changed significantly in the roots of C. tagal under salt shock, of which 83 had a more than 2-fold change and were further assembled into 59 unigenes. Global transcription was stable at the early stage of salt stress and then was gradually dysregulated with the increased duration of the stress. Importantly, a pair-wise comparison of predicted homologous gene pairs revealed that the transcriptional regulations of most of the differentially expressed genes were highly divergent in C. tagal from that in salt-sensitive species, Arabidopsis thaliana. Conclusions/Significance This work suggests that transcriptional homeostasis and specific transcriptional regulation are major events in the roots of C. tagal when subjected to salt shock, which could contribute to the establishment of adaptation to saline environments and, thus, facilitate the salt-tolerant lifestyle of this mangrove species. Furthermore, the candidate genes underlying the adaptation were identified through comparative analyses. This study provides a foundation for dissecting the genetic basis of the adaptation of mangroves to intertidal environments. PMID:22574172

  3. Microarray analysis of microRNA expression during axolotl limb regeneration.

    PubMed

    Holman, Edna C; Campbell, Leah J; Hines, John; Crews, Craig M

    2012-01-01

    Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and