ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

USDA-ARS?s Scientific Manuscript database

We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

eSensor®: A Microarray Technology Based on Electrochemical Detection of Nucleic Acids and Its Application to Cystic Fibrosis Carrier Screening

NASA Astrophysics Data System (ADS)

Reed, Michael R.; Coty, William A.

We have developed a test for identification of carriers for cystic fibrosis using the eSensor® DNA detection technology. Oligonucleotide probes are deposited within self-assembled monolayers on gold electrodes arrayed upon printed circuit boards. These probes allow sequence-specific capture of amplicons containing a panel of mutation sites associated with cystic fibrosis. DNA targets are detected and mutations genotyped using a “sandwich” assay methodology employing electrochemical detection of ferrocene-labeled oligonucleotides for discrimination of carrier and non-carrier alleles. Performance of the cystic fibrosis application demonstrates sufficient accuracy and reliability for clinical diagnostic use, and the procedure can be performed by trained medical technologists available in the hospital laboratory.

Design of oligonucleotides for microarrays and perspectives for design of multi-transcriptome arrays.

PubMed

Nielsen, Henrik Bjørn; Wernersson, Rasmus; Knudsen, Steen

2003-07-01

Optimal design of oligonucleotides for microarrays involves tedious and laborious work evaluating potential oligonucleotides relative to a series of parameters. The currently available tools for this purpose are limited in their flexibility and do not present the oligonucleotide designer with an overview of these parameters. We present here a flexible tool named OligoWiz for designing oligonucleotides for multiple purposes. OligoWiz presents a set of parameter scores in a graphical interface to facilitate an overview for the user. Additional custom parameter scores can easily be added to the program to extend the default parameters: homology, DeltaTm, low-complexity, position and GATC-only. Furthermore we present an analysis of the limitations in designing oligonucleotide sets that can detect transcripts from multiple organisms. OligoWiz is available at www.cbs.dtu.dk/services/OligoWiz/.

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. © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-05-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication

PubMed Central

Moorcroft, Matthew J.; Meuleman, Wouter R. A.; Latham, Steven G.; Nicholls, Thomas J.; Egeland, Ryan D.; Southern, Edwin M.

2005-01-01

In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 μm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling. High, reproducible yields were achieved for both 6mer and 21mer probes as assessed by hybridization to fluorescent oligonucleotides. Oligonucleotide surface density was comparable with that obtained on glass substrates. These results suggest PDMS as a stable and flexible alternative to glass as a suitable substrate in the fabrication and synthesis of DNA microarrays. PMID:15870385

Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

DOEpatents

Golova, Julia B.; Chernov, Boris K.

2010-04-27

New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

DNA Microarray-based Ecotoxicological Biomarker Discovery in a Small Fish Model Species

EPA Science Inventory

This paper addresses several issues critical to use of zebrafish oligonucleotide microarrays for computational toxicology research on endocrine disrupting chemicals using small fish models, and more generally, the use of microarrays in aquatic toxicology.

A DNA microarray for identification of selected Korean birds based on mitochondrial cytochrome c oxidase I gene sequences.

PubMed

Chung, In-Hyuk; Yoo, Hye Sook; Eah, Jae-Yong; Yoon, Hyun-Kyu; Jung, Jin-Wook; Hwang, Seung Yong; Kim, Chang-Bae

2010-10-01

DNA barcoding with the gene encoding cytochrome c oxidase I (COI) in the mitochondrial genome has been proposed as a standard marker to identify and discover animal species. Some migratory wild birds are suspected of transmitting avian influenza and pose a threat to aircraft safety because of bird strikes. We have previously reported the COI gene sequences of 92 Korean bird species. In the present study, we developed a DNA microarray to identify 17 selected bird species on the basis of nucleotide diversity. We designed and synthesized 19 specific oligonucleotide probes; these probes were arrayed on a silylated glass slide. The length of the probes was 19-24 bps. The COI sequences amplified from the tissues of the selected birds were labeled with a fluorescent probe for microarray hybridization, and unique hybridization patterns were detected for each selected species. These patterns may be considered diagnostic patterns for species identification. This microarray system will provide a sensitive and a high-throughput method for identification of Korean birds.

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

PubMed Central

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

2005-01-01

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

Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

NASA Technical Reports Server (NTRS)

El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

2003-01-01

Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

PubMed Central

Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

2005-01-01

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

PubMed Central

2013-01-01

Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays

PubMed Central

2013-01-01

Background Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. Results We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. Conclusions Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different surfaces in dependence of the applied spotting and reaction volume. PMID:23758982

Universal Oligonucleotide Microarray for Sub-Typing of Influenza A Virus

PubMed Central

Ryabinin, Vladimir A.; Kostina, Elena V.; Maksakova, Galiya A.; Neverov, Alexander A.; Chumakov, Konstantin M.; Sinyakov, Alexander N.

2011-01-01

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1–H13, H15, H16) and neuraminidase (N1–N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus. PMID:21559081

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

PubMed

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

2014-08-01

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

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

PubMed Central

LeProust, Emily M.; Peck, Bill J.; Spirin, Konstantin; McCuen, Heather Brummel; Moore, Bridget; Namsaraev, Eugeni; Caruthers, Marvin H.

2010-01-01

We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies’ SurePrint® DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology). PMID:20308161

DEVELOPMENT AND VALIDATION OF A 2,000 GENE MICROARRAY FOR THE FATHEAD MINNOW, PIMEPHALES PROMELAS

EPA Science Inventory

The development of the gene microarray has provided the field of ecotoxicology a new tool to identify modes of action (MOA) of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000 gene oligonucleotide microarray for the fathead minnow (P...

nuID: a universal naming scheme of oligonucleotides for Illumina, Affymetrix, and other microarrays

PubMed Central

Du, Pan; Kibbe, Warren A; Lin, Simon M

2007-01-01

Background Oligonucleotide probes that are sequence identical may have different identifiers between manufacturers and even between different versions of the same company's microarray; and sometimes the same identifier is reused and represents a completely different oligonucleotide, resulting in ambiguity and potentially mis-identification of the genes hybridizing to that probe. Results We have devised a unique, non-degenerate encoding scheme that can be used as a universal representation to identify an oligonucleotide across manufacturers. We have named the encoded representation 'nuID', for nucleotide universal identifier. Inspired by the fact that the raw sequence of the oligonucleotide is the true definition of identity for a probe, the encoding algorithm uniquely and non-degenerately transforms the sequence itself into a compact identifier (a lossless compression). In addition, we added a redundancy check (checksum) to validate the integrity of the identifier. These two steps, encoding plus checksum, result in an nuID, which is a unique, non-degenerate, permanent, robust and efficient representation of the probe sequence. For commercial applications that require the sequence identity to be confidential, we have an encryption schema for nuID. We demonstrate the utility of nuIDs for the annotation of Illumina microarrays, and we believe it has universal applicability as a source-independent naming convention for oligomers. Reviewers This article was reviewed by Itai Yanai, Rong Chen (nominated by Mark Gerstein), and Gregory Schuler (nominated by David Lipman). PMID:17540033

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

PubMed

Bae, Jin-Woo; Park, Yong-Ha

2006-07-01

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

Remediation of petroleum hydrocarbon-contaminated sites by DNA diagnosis-based bioslurping technology.

PubMed

Kim, Seungjin; Krajmalnik-Brown, Rosa; Kim, Jong-Oh; Chung, Jinwook

2014-11-01

The application of effective remediation technologies can benefit from adequate preliminary testing, such as in lab-scale and Pilot-scale systems. Bioremediation technologies have demonstrated tremendous potential with regards to cost, but they cannot be used for all contaminated sites due to limitations in biological activity. The purpose of this study was to develop a DNA diagnostic method that reduces the time to select contaminated sites that are good candidates for bioremediation. We applied an oligonucleotide microarray method to detect and monitor genes that lead to aliphatic and aromatic degradation. Further, the bioremediation of a contaminated site, selected based on the results of the genetic diagnostic method, was achieved successfully by applying bioslurping in field tests. This gene-based diagnostic technique is a powerful tool to evaluate the potential for bioremediation in petroleum hydrocarbon contaminated soil. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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

2003-11-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-02-19

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

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

PubMed Central

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

2002-01-01

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

Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer.

PubMed

Beyer, Sasha J; Zhang, Xiaoli; Jimenez, Rafael E; Lee, Mei-Ling T; Richardson, Andrea L; Huang, Kun; Jhiang, Sissy M

2011-10-11

Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

sigReannot: an oligo-set re-annotation pipeline based on similarities with the Ensembl transcripts and Unigene clusters.

PubMed

Casel, Pierrot; Moreews, François; Lagarrigue, Sandrine; Klopp, Christophe

2009-07-16

Microarray is a powerful technology enabling to monitor tens of thousands of genes in a single experiment. Most microarrays are now using oligo-sets. The design of the oligo-nucleotides is time consuming and error prone. Genome wide microarray oligo-sets are designed using as large a set of transcripts as possible in order to monitor as many genes as possible. Depending on the genome sequencing state and on the assembly state the knowledge of the existing transcripts can be very different. This knowledge evolves with the different genome builds and gene builds. Once the design is done the microarrays are often used for several years. The biologists working in EADGENE expressed the need of up-to-dated annotation files for the oligo-sets they share including information about the orthologous genes of model species, the Gene Ontology, the corresponding pathways and the chromosomal location. The results of SigReannot on a chicken micro-array used in the EADGENE project compared to the initial annotations show that 23% of the oligo-nucleotide gene annotations were not confirmed, 2% were modified and 1% were added. The interest of this up-to-date annotation procedure is demonstrated through the analysis of real data previously published. SigReannot uses the oligo-nucleotide design procedure criteria to validate the probe-gene link and the Ensembl transcripts as reference for annotation. It therefore produces a high quality annotation based on reference gene sets.

Oligonucleotide microarray for subtyping of influenza A viruses

NASA Astrophysics Data System (ADS)

Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

2012-02-01

Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

PubMed Central

2011-01-01

Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

An Undergraduate Laboratory Exercise to Study the Effect of Darkness on Plant Gene Expression Using DNA Microarray

ERIC Educational Resources Information Center

Chang, Ming-Mei; Briggs, George M.

2007-01-01

DNA microarrays are microscopic arrays on a solid surface, typically a glass slide, on which DNA oligonucleotides are deposited or synthesized in a high-density matrix with a predetermined spatial order. Several types of DNA microarrays have been developed and used for various biological studies. Here, we developed an undergraduate laboratory…

Oligo Design: a computer program for development of probes for oligonucleotide microarrays.

PubMed

Herold, Keith E; Rasooly, Avraham

2003-12-01

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

ASSESSING THE USE OF OLIGONUCLEOTIDE MICROARRAYS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) TO EXAMINE EXPOSURE VARIABLES

EPA Science Inventory

Microarray technology has proven to be a useful tool for analyzing the transcriptome of various organisms representing conditions such as disease states, developmental stages, and responses to chemical exposure. Although most commercially available arrays are limited to organism...

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

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

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

2000-01-01

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

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

PubMed Central

2011-01-01

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

Methylation oligonucleotide microarray: a novel tool to analyze methylation patterns

NASA Astrophysics Data System (ADS)

Hou, Peng; Ji, Meiju; He, Nongyao; Lu, Zuhong

2003-04-01

A new technique to analyze methylation patterns in several adjacent CpG sites was developed and reported here. We selected a 336bp segment of the 5"-untranslated region and the first exon of the p16Ink4a gene, which include the most densely packed CpG fragment of the islands containing 32 CpG dinucleotides, as the investigated target. The probes that include all types of methylation patterns were designed to fabricate a DNA microarray to determine the methylation patterns of seven adjacent CpG dinucleotides sites. High accuracy and reproducibility were observed in several parallel experiments. The results led us to the conclusion that the methylation oligonucleotide microarray can be applied as a novel and powerful tool to map methylation patterns and changes in multiple CpG island loci in a variety of tumors.

Particle-Based Microarrays of Oligonucleotides and Oligopeptides.

PubMed

Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F Ralf; Breitling, Frank; Loeffler, Felix F

2014-10-28

In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

Particle-Based Microarrays of Oligonucleotides and Oligopeptides

PubMed Central

Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K.; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F. Ralf; Breitling, Frank; Loeffler, Felix F.

2014-01-01

In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches. PMID:27600347

Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges

PubMed Central

Naiser, Thomas; Ehler, Oliver; Kayser, Jona; Mai, Timo; Michel, Wolfgang; Ott, Albrecht

2008-01-01

Background The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations. Results We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form). Conclusion We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. PMID:18477387

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

PubMed

Elsholz, B; Wörl, R; Blohm, L; Albers, J; Feucht, H; Grunwald, T; Jürgen, B; Schweder, T; Hintsche, Rainer

2006-07-15

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

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

EPA Science Inventory

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

Chromosomal microarray in clinical diagnosis: a study of 337 patients with congenital anomalies and developmental delays or intellectual disability.

PubMed

Sansović, Ivona; Ivankov, Ana-Maria; Bobinec, Adriana; Kero, Mijana; Barišić, Ingeborg

2017-06-14

To determine the diagnostic yield and criteria that could help to classify and interpret the copy number variations (CNVs) detected by chromosomal microarray (CMA) technique in patients with congenital and developmental abnormalities including dysmorphia, developmental delay (DD) or intellectual disability (ID), autism spectrum disorders (ASD) and congenital anomalies (CA). CMA analysis was performed in 337 patients with DD/ID with or without dysmorphism, ASD, and/or CA. In 30 of 337 patients, chromosomal imbalances had previously been detected by classical cytogenetic and molecular cytogenetic methods. In 73 of 337 patients, clinically relevant variants were detected and better characterized. Most of them were >1 Mb. Variants of unknown clinical significance (VOUS) were discovered in 35 patients. The most common VOUS size category was <300 kb (40.5%). Deletions and de novo imbalances were more frequent in pathogenic CNV than in VOUS category. CMA had a high diagnostic yield of 43/307, excluding patients previously detected by other methods. CMA was valuable in establishing the diagnosis in a high proportion of patients. Criteria for classification and interpretation of CNVs include CNV size and type, mode of inheritance, and genotype-phenotype correlation. Agilent ISCA v2 Human Genome 8x60 K oligonucleotide microarray format proved to be reasonable resolution for clinical use, particularly in the regions that are recommended by the International Standard Cytogenomic Array (ISCA) Consortium and associated with well-established syndromes.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

PubMed

Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

2015-06-01

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Microarray analysis identified Puccinia striiformis f. sp. tritici genes involved in infection and sporulation.

USDA-ARS?s Scientific Manuscript database

Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, one of the most important diseases of wheat worldwide. To identify Pst genes involved in infection and sporulation, a custom oligonucleotide Genechip was made using sequences of 442 genes selected from Pst cDNA libraries. Microarray analy...

A Graphical Systems Model and Tissue-specific Functional Gene Sets to Aid Transcriptomic Analysis of Chemical Impacts on the Female Teleost Reproductive Axis

EPA Science Inventory

Oligonucleotide microarrays and other ‘omics’ approaches are powerful tools for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the poor power of microarray-b...

Spotting and validation of a genome wide oligonucleotide chip with duplicate measurement of each gene

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

Thomassen, Mads; Skov, Vibe; Eiriksdottir, Freyja

2006-06-16

The quality of DNA microarray based gene expression data relies on the reproducibility of several steps in a microarray experiment. We have developed a spotted genome wide microarray chip with oligonucleotides printed in duplicate in order to minimise undesirable biases, thereby optimising detection of true differential expression. The validation study design consisted of an assessment of the microarray chip performance using the MessageAmp and FairPlay labelling kits. Intraclass correlation coefficient (ICC) was used to demonstrate that MessageAmp was significantly more reproducible than FairPlay. Further examinations with MessageAmp revealed the applicability of the system. The linear range of the chips wasmore » three orders of magnitude, the precision was high, as 95% of measurements deviated less than 1.24-fold from the expected value, and the coefficient of variation for relative expression was 13.6%. Relative quantitation was more reproducible than absolute quantitation and substantial reduction of variance was attained with duplicate spotting. An analysis of variance (ANOVA) demonstrated no significant day-to-day variation.« less

Oligonucleotide microarray chip for the quantification of MS2, ΦX174, and adenoviruses on the multiplex analysis platform MCR 3.

PubMed

Lengger, Sandra; Otto, Johannes; Elsässer, Dennis; Schneider, Oliver; Tiehm, Andreas; Fleischer, Jens; Niessner, Reinhard; Seidel, Michael

2014-05-01

Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4-4.5 h. The parallel quantification was possible in a concentration range of 9.6 × 10(5)-1.4 × 10(10) genomic units (GU)/mL for bacteriophage MS2, 1.4 × 10(5)-3.7 × 10(8) GU/mL for bacteriophage ΦX174, and 6.5 × 10(3)-1.2 × 10(5) for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6 × 10(5) GU/mL for MS2, 5.3 × 10(3) GU/mL for ΦX174, and 1.5 × 10(2) GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.

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

PubMed

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

2015-07-01

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

Nucleic acid sequence detection using multiplexed oligonucleotide PCR

DOEpatents

Nolan, John P [Santa Fe, NM; White, P Scott [Los Alamos, NM

2006-12-26

Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides.

PubMed

Laassri, Majid; Chizhikov, Vladimir; Mikheev, Maxim; Shchelkunov, Sergei; Chumakov, Konstantin

2003-09-01

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

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

PubMed

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

2006-09-01

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

Development and assessment of whole-genome oligonucleotide microarrays to analyze an anaerobic microbial community and its responses to oxidative stress.

PubMed

Scholten, Johannes C M; Culley, David E; Nie, Lei; Munn, Kyle J; Chow, Lely; Brockman, Fred J; Zhang, Weiwen

2007-06-29

The application of DNA microarray technology to investigate multiple-species microbial communities presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri, and Methanospirillum hungatei, and their application to analyze global gene expression in a four-species microbial community in response to oxidative stress. In order to minimize the possibility of cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. This study showed that S. fumaroxidans and M. hungatei responded to the oxidative stress with up-regulation of several genes known to be involved in reactive oxygen species (ROS) detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. However, D. vulgaris seemed to be less sensitive to the oxidative stress as a member of a four-species community, since no gene involved in ROS detoxification was up-regulated. Our work demonstrated the successful application of microarrays to a multiple-species microbial community, and our preliminary results indicated that this approach could provide novel insights on the metabolism within microbial communities.

Double stranded nucleic acid biochips

DOEpatents

Chernov, Boris; Golova, Julia

2006-05-23

This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

NASA Technical Reports Server (NTRS)

Urakawa, Hidetoshi; El Fantroussi, Said; Smidt, Hauke; Smoot, James C.; Tribou, Erik H.; Kelly, John J.; Noble, Peter A.; Stahl, David A.

2003-01-01

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

Ethical issues raised by genetic testing with oligonucleotide microarrays.

PubMed

Grody, Wayne W

2003-02-01

Because genes and alterations within them determine the identity, characteristics, and inheritance of every individual, the application of genetic science to humans has long been surrounded by apprehension, controversy, and real or perceived potential for abuse. Crude eugenics practices of the past now find a theoretical rebirth and transformation through the use of modern molecular genetic technologies for mutation detection, predictive and prenatal diagnosis, and, ultimately, gene replacement. The advent of oligonucleotide microarray analysis, in which hundreds or thousands of genes and mutations can be tested in parallel, offers tremendous promise for more accurate, sensitive, and efficient genetic testing. At the same time, however, this powerful technology dramatically increases the number and scope of ethical concerns accompanying each individual test request. This article considers the evolution and implications of these concerns, from the initial ordering of a microarray test by the physician to such issues as informed consent, privacy, confidentiality, clinical utility, discrimination, stigmatization, ethnic and population impact, and reimbursement.

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.

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

USDA-ARS?s Scientific Manuscript database

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

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

PubMed Central

2013-01-01

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms. PMID:24160452

Soft Lithography for Oligonucleotide Arrays Fabrication

DTIC Science & Technology

2001-10-25

adenosine; Abbreviated T, C, G, A respectively), the other synthesis reagents and solvents except oxidation agent (seen in Table 1) were purchased...dried by cold blowing before hybridization. Oligonucleotide arrays were hybridized in 200 nM 3’-TCC TCC GAT TCA GAG AGT CC- HEX (PE Biosystems... citrate buffer), 0.1% SDS in 0.1xSSC respectively. The probe array was scanned on the Scanarray Microarray Systems (Packard Biochip Technologies, USA

Selection of optimal oligonucleotide probes for microarrays usingmultiple criteria, global alignment and parameter estimation.

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

Li, Xingyuan; He, Zhili; Zhou, Jizhong

2005-10-30

The oligonucleotide specificity for microarray hybridizationcan be predicted by its sequence identity to non-targets, continuousstretch to non-targets, and/or binding free energy to non-targets. Mostcurrently available programs only use one or two of these criteria, whichmay choose 'false' specific oligonucleotides or miss 'true' optimalprobes in a considerable proportion. We have developed a software tool,called CommOligo using new algorithms and all three criteria forselection of optimal oligonucleotide probes. A series of filters,including sequence identity, free energy, continuous stretch, GC content,self-annealing, distance to the 3'-untranslated region (3'-UTR) andmelting temperature (Tm), are used to check each possibleoligonucleotide. A sequence identity is calculated based onmore » gapped globalalignments. A traversal algorithm is used to generate alignments for freeenergy calculation. The optimal Tm interval is determined based on probecandidates that have passed all other filters. Final probes are pickedusing a combination of user-configurable piece-wise linear functions andan iterative process. The thresholds for identity, stretch and freeenergy filters are automatically determined from experimental data by anaccessory software tool, CommOligo_PE (CommOligo Parameter Estimator).The program was used to design probes for both whole-genome and highlyhomologous sequence data. CommOligo and CommOligo_PE are freely availableto academic users upon request.« less

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

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

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

2006-10-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

Towards MRI microarrays.

PubMed

Hall, Andrew; Mundell, Victoria J; Blanco-Andujar, Cristina; Bencsik, Martin; McHale, Glen; Newton, Michael I; Cave, Gareth W V

2010-04-14

Superparamagnetic iron oxide nanometre scale particles have been utilised as contrast agents to image staked target binding oligonucleotide arrays using MRI to correlate the signal intensity and T(2)* relaxation times in different NMR fluids.

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

PubMed

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

2016-03-01

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

Curation of microarray oligonucleotides and corresponding ESTs/cDNAs used for gene expression analysis in zebra finches.

PubMed

Lovell, Peter V; Huizinga, Nicole A; Getachew, Abel; Mees, Brianna; Friedrich, Samantha R; Wirthlin, Morgan; Mello, Claudio V

2018-05-18

Zebra finches are a major model organism for investigating mechanisms of vocal learning, a trait that enables spoken language in humans. The development of cDNA collections with expressed sequence tags (ESTs) and microarrays has allowed for extensive molecular characterizations of circuitry underlying vocal learning and production. However, poor database curation can lead to errors in transcriptome and bioinformatics analyses, limiting the impact of these resources. Here we used genomic alignments and synteny analysis for orthology verification to curate and reannotate ~ 35% of the oligonucleotides and corresponding ESTs/cDNAs that make-up Agilent microarrays for gene expression analysis in finches. We found that: (1) 5475 out of 43,084 oligos (a) failed to align to the zebra finch genome, (b) aligned to multiple loci, or (c) aligned to Chr_un only, and thus need to be flagged until a better genome assembly is available, or (d) reflect cloning artifacts; (2) Out of 9635 valid oligos examined further, 3120 were incorrectly named, including 1533 with no known orthologs; and (3) 2635 oligos required name update. The resulting curated dataset provides a reference for correcting gene identification errors in previous finch microarrays studies, and avoiding such errors in future studies.

EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments.

PubMed

Milewski, Marek C; Kamel, Karol; Kurzynska-Kokorniak, Anna; Chmielewski, Marcin K; Figlerowicz, Marek

2017-10-01

Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

PubMed

Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

2012-01-01

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

Development of the first oligonucleotide microarray for global gene expression profiling in guinea pigs: defining the transcription signature of infectious diseases.

PubMed

Jain, Ruchi; Dey, Bappaditya; Tyagi, Anil K

2012-10-02

The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model. By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection. We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

Replication dynamics of the yeast genome.

PubMed

Raghuraman, M K; Winzeler, E A; Collingwood, D; Hunt, S; Wodicka, L; Conway, A; Lockhart, D J; Davis, R W; Brewer, B J; Fangman, W L

2001-10-05

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

Development of a genotyping microarray for Usher syndrome.

PubMed

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner-Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva-Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-02-01

Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

Development of a genotyping microarray for Usher syndrome

PubMed Central

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner‐Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva‐Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-01-01

Background Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein‐coding exons. Methods: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele‐specific oligonucleotides corresponding to all 298 Usher syndrome‐associated sequence variants known to date, 76 of which are novel, were arrayed. Results Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. Conclusion The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first‐pass screening tool. PMID:16963483

Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

PubMed

Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

2016-04-01

Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

PubMed

Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W; Gallardo-Escarate, Cristian; Planas, Josep V; Mackenzie, Simon

2017-02-03

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

PubMed Central

Narihiro, Takashi; Sekiguchi, Yuji

2011-01-01

Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide

PubMed Central

Hwang, Byungjin; Bang, Duhee

2016-01-01

All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials. PMID:27876825

Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide.

PubMed

Hwang, Byungjin; Bang, Duhee

2016-11-23

All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials.

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

PubMed Central

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

2013-01-01

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

PCR amplification on microarrays of gel immobilized oligonucleotides

DOEpatents

Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

2003-11-04

The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

An oligonucleotide microarray to characterize multidrug resistant plasmids

USDA-ARS?s Scientific Manuscript database

Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the host organism like antibiotic drug resistance. Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replic...

Linear model for fast background subtraction in oligonucleotide microarrays.

PubMed

Kroll, K Myriam; Barkema, Gerard T; Carlon, Enrico

2009-11-16

One important preprocessing step in the analysis of microarray data is background subtraction. In high-density oligonucleotide arrays this is recognized as a crucial step for the global performance of the data analysis from raw intensities to expression values. We propose here an algorithm for background estimation based on a model in which the cost function is quadratic in a set of fitting parameters such that minimization can be performed through linear algebra. The model incorporates two effects: 1) Correlated intensities between neighboring features in the chip and 2) sequence-dependent affinities for non-specific hybridization fitted by an extended nearest-neighbor model. The algorithm has been tested on 360 GeneChips from publicly available data of recent expression experiments. The algorithm is fast and accurate. Strong correlations between the fitted values for different experiments as well as between the free-energy parameters and their counterparts in aqueous solution indicate that the model captures a significant part of the underlying physical chemistry.

SINGLE-BASE-PAIR MISMATCH DISCRIMINATION USING OLIGONUCLEOTIDE DNA MICROARRAYS AND MELTING PROFILES. (R829458C004)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

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

Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection inmore » archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.« less

Microarray Genomic Systems Development

DTIC Science & Technology

2008-06-01

11 species), Escherichia coli TOP10 (7 strains), and Geobacillus stearothermophilus . Using standard molecular biology methods, we isolated genomic...comparisons. Results: Different species of bacteria, including Escherichia coli, Bacillus bacteria, and Geobacillus stearothermophilus produce qualitatively...oligonucleotides to labelled genomic DNA from a set of test samples, including eleven Bacillus species, Geobacillus stearothermophilus , and seven Escherichia

Isolation of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays - Application in Aerospace Medicine

DTIC Science & Technology

2004-01-01

of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays  Application in...VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND KINETIC RT-PCR ASSAYS  APPLICATION IN AEROSPACE MEDICINE INTRODUCTION Extraction of cellular

High-density fiber optic biosensor arrays

NASA Astrophysics Data System (ADS)

Epstein, Jason R.; Walt, David R.

2002-02-01

Novel approaches are required to coordinate the immense amounts of information derived from diverse genomes. This concept has influenced the expanded role of high-throughput DNA detection and analysis in the biological sciences. A high-density fiber optic DNA biosensor was developed consisting of oligonucleotide-functionalized, 3.1 mm diameter microspheres deposited into the etched wells on the distal face of a 500 micrometers imaging fiber bundle. Imaging fiber bundles containing thousands of optical fibers, each associated with a unique oligonucleotide probe sequence, were the foundation for an optically connected, individually addressable DNA detection platform. Different oligonucleotide-functionalized microspheres were combined in a stock solution, and randomly dispersed into the etched wells. Microsphere positions were registered from optical dyes incorporated onto the microspheres. The distribution process provided an inherent redundancy that increases the signal-to-noise ratio as the square root of the number of sensors examined. The representative amount of each probe-type in the array was dependent on their initial stock solution concentration, and as other sequences of interest arise, new microsphere elements can be added to arrays without altering the existing detection capabilities. The oligonucleotide probe sequences hybridize to fluorescently-labeled, complementary DNA target solutions. Fiber optic DNA microarray research has included DNA-protein interaction profiles, microbial strain differentiation, non-labeled target interrogation with molecular beacons, and single cell-based assays. This biosensor array is proficient in DNA detection linked to specific disease states, single nucleotide polymorphism (SNP's) discrimination, and gene expression analysis. This array platform permits multiple detection formats, provides smaller feature sizes, and enables sensor design flexibility. High-density fiber optic microarray biosensors provide a fast, reversible format with the detection limit of a few hundred molecules.

High-density, microsphere-based fiber optic DNA microarrays.

PubMed

Epstein, Jason R; Leung, Amy P K; Lee, Kyong Hoon; Walt, David R

2003-05-01

A high-density fiber optic DNA microarray has been developed consisting of oligonucleotide-functionalized, 3.1-microm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle. The fiber bundles are comprised of 6000-50000 fused optical fibers and each fiber terminates with an etched well. The microwell array is capable of housing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. The array fabrication process results in random microsphere placement. Determining the position of microspheres in the random array requires an optical encoding scheme. This array platform provides many advantages over other array formats. The microsphere-stock suspension concentration added to the etched fiber can be controlled to provide inherent sensor redundancy. Examining identical microspheres has a beneficial effect on the signal-to-noise ratio. As other sequences of interest are discovered, new microsphere sensing elements can be added to existing microsphere pools and new arrays can be fabricated incorporating the new sequences without altering the existing detection capabilities. These microarrays contain the smallest feature sizes (3 microm) of any DNA array, allowing interrogation of extremely small sample volumes. Reducing the feature size results in higher local target molecule concentrations, creating rapid and highly sensitive assays. The microsphere array platform is also flexible in its applications; research has included DNA-protein interaction profiles, microbial strain differentiation, and non-labeled target interrogation with molecular beacons. Fiber optic microsphere-based DNA microarrays have a simple fabrication protocol enabling their expansion into other applications, such as single cell-based assays.

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.

Application of HLA-DRB1 genotyping by oligonucleotide micro-array technology in forensic medicine.

PubMed

Jiang, Bin; Li, Yao; Wu, Hai; He, Xianmin; Li, Chengtao; Li, Li; Tang, Rong; Xie, Yi; Mao, Yumin

2006-10-16

The human leukocyte antigen (HLA) system is known to be the most complex polymorphic system in the human genome. Among all of the HLA loci, HLA-DRB1 has the second largest number of alleles. The purpose of this study is to develop an oligonucleotide micro-array based HLA-DRB1 typing system for use in forensic identification, anthropology, tissue transplantation, and other genetic research fields. The system was developed by analyzing the HLA-DRB1 (DRB1) genotypes in 1198 unrelated healthy Chinese Han individuals originating from various parts of China and residing in Shanghai, China. Polymerase chain reaction (PCR) coupled with the oligonucleotide micro-array technology was used to detect and type HLA-DRB1 alleles of the sample individuals. The reliability, sensitivity, consistency and specificity were evaluated for use in forensic identification. Furthermore, a meta-analysis was carried out by comparing the allele frequencies of the HLA-DRB1 locus with those of other Chinese Han groups, Chinese minorities and other ethnic populations. All the DNA samples yielded a 273 bp amplification product, with no other amplification products in this length range. The minimum quantity of DNA detected by this method is 15 ng in a PCR reaction system of 25 microl. The population studied appeared to be not in Hardy-Weinberg equilibrium. Observed heterozygosity (Ho), expected heterozygosity (He), expected probability of exclusion (PE), polymorphic information content (PIC), and discrimination power (DP) of the HLA-DRB1 locus from the Shanghai Han ethnic group were evaluated to be 0.8022, 0.8870, 0.7741, 0.8771, 0.9750, respectively. A total of 25 HLA-DRB1 alleles were identified. HLA-DRB1*09XX, *04XX, *12XX and *15XX were the most frequent DRB1 alleles, which were observed in 58.76% of the sample. One hundred and sixteen genotypes were found. The five most frequent genotypes were: *04XX/*04XX (0.0626), *09XX/*09XX (0.0593), *04XX/*09XX (0.0551), *09XX/*15XX (0.0384) and *08XX/*12XX (0.0351). The meta-analysis showed that there were uniquely distributed features of DRB1 alleles among various ethnic populations and among the studied population groups from various regions with the same ethnic origin. An HLA-DRB1 genotyping system has been developed and established based on the oligonucleotide micro-array technology. The HLA-DRB1 typing of the Han population in Shanghai has revealed a relatively high heterogeneity. Information obtained in this study will be useful for medical and forensic applications as well as in anthropology research. Large-scale micro-array detection is highly accurate and reliable for DNA-based HLA-DRB1 genotyping. These results suggest that HLA-DRB1 DNA polymorphisms and the database of the Shanghai Han group have useful applications in processing forensic casework (as personal identification, paternity test), tracing population migration and genetic diagnosis.

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

PubMed

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

2004-06-01

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

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

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

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

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

PubMed

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

2013-07-01

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

PubMed Central

Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

2013-01-01

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

[Differential gene expression in incompatible interaction between Lilium regale Wilson and Fusarium oxysporum f. sp. lilii revealed by combined SSH and microarray analysis].

PubMed

Rao, J; Liu, D; Zhang, N; He, H; Ge, F; Chen, C

2014-01-01

Fusarium wilt, caused by a soilborne pathogen Fusarium oxysporum f. sp. lilii, is the major disease of lily (Lilium L.). In order to isolate the genes differentially expressed in a resistant reaction to F. oxysporum in L. regale Wilson, a cDNA library was constructed with L. regale root during F. oxysporum infection using the suppression subtractive hybridization (SSH), and a total of 585 unique expressed sequence tags (ESTs) were obtained. Furthermore, the gene expression profiles in the incompatible interaction between L. regale and F. oxysporum were revealed by oligonucleotide microarray analysis of 585 unique ESTs comparison to the compatible interaction between a susceptible Lilium Oriental Hybrid 'Siberia' and F. oxysporum. The result of expression profile analysis indicated that the genes encoding pathogenesis-related proteins (PRs), antioxidative stress enzymes, secondary metabolism enzymes, transcription factors, signal transduction proteins as well as a large number of unknown genes were involved in early defense response of L. regale to F. oxysporum infection. Moreover, the following quantitative reverse transcription PCR (QRT-PCR) analysis confirmed reliability of the oligonucleotide microarray data. In the present study, isolation of differentially expressed genes in L. regale during response to F. oxysporum helped to uncover the molecular mechanism associated with the resistance of L. regale against F. oxysporum.

Use of DNA Microarrays to Identify Diagnostic Signature Transcription Profiles for Host Responses to Infectious Agents

DTIC Science & Technology

2004-10-01

informative in this regard. Key signature genes will serve as the basis for rapid diagnostic approaches that could be accessed when an outbreak is suspected...AD Award Number: DAMD17-01-1-0787 TITLE: Use of DNA Microarrays to Identify Diagnostic Signature Transcription Profiles for Host Responses to...Sep 2004) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Use of DNA Microarrays to Identify Diagnostic Signature DAMD17-01-1-0787 Transcription Profiles for

Differences in X-chromosome transcriptional activity and cholesterol metabolism between placentae from swine breeds from asian and western origins

USDA-ARS?s Scientific Manuscript database

To gain insight into placental physiology differences between the Chinese Meishan and white composite (WC) swine breeds, short-oligonucleotide microarray gene expression profiles of gestational Day 25, 45, 65, 85, and 105 placental tissues were compared. Differential expression was determined by a ...

Analysis of large-scale gene expression data.

PubMed

Sherlock, G

2000-04-01

The advent of cDNA and oligonucleotide microarray technologies has led to a paradigm shift in biological investigation, such that the bottleneck in research is shifting from data generation to data analysis. Hierarchical clustering, divisive clustering, self-organizing maps and k-means clustering have all been recently used to make sense of this mass of data.

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

Andersen, G.L.; He, Z.; DeSantis, T.Z.

Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogeneticmore » microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.« less

Oligonucleotide microarray analysis reveals dysregulation of energy-related metabolism in insulin-sensitive tissues of type 2 diabetes patients.

PubMed

Wang, M; Wang, X C; Zhao, L; Zhang, Y; Yao, L L; Lin, Y; Peng, Y D; Hu, R M

2014-06-17

Impaired insulin action within skeletal muscle, adipose tissue, and the liver is an important characteristic of type 2 diabetes (T2D). In order to identify common underlying defects in insulin-sensitive tissues that may be involved in the pathogenesis of T2D, the gene expression profiles of skeletal muscle, visceral adipose tissue, and liver from autopsy donors with or without T2D were examined using oligonucleotide microarrays and quantitative reverse transcriptase-PCR. Compared with controls, 691 genes were commonly dysregulated in these three insulin-sensitive tissues of humans with T2D. These co-expressed genes were enriched within the mitochondrion, with suggested involvement in energy metabolic processes such as glycolysis and gluconeogenesis, fatty acid beta oxidative, tricarboxylic acid cycle, and electron transport. Genes related to energy metabolism were mostly downregulated in diabetic skeletal muscle and visceral adipose tissue, while they were upregulated in the diabetic liver. This observed dysregulation in energy-related metabolism may be the underlying factor leading to the molecular mechanisms responsible for the insulin resistance of patients with T2D.

Regulation of the activity of the promoter of RNA-induced silencing, C3PO.

PubMed

Sahu, Shriya; Williams, Leo; Perez, Alberto; Philip, Finly; Caso, Giuseppe; Zurawsky, Walter; Scarlata, Suzanne

2017-09-01

RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (component 3 of RISC). We have previously found that phospholipase Cβ, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes. Understanding the parameters that control C3PO activity and which genes are impacted by G protein activation would help predict which genes are more vulnerable to downregulation. Here, using a library of 10 18 oligonucleotides, we show that C3PO binds oligonucleotides with structural specificity but little sequence specificity. Alternately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to substrate stability. Importantly, we find that oligonucleotides with higher Tm values are inhibited by bound PLCβ. This finding is supported by microarray analysis in cells over-expressing PLCβ1. Taken together, this study allows predictions of the genes whose post-transcriptional regulation is responsive to the G protein/phospholipase Cβ/calcium signaling pathway. © 2017 The Protein Society.

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

PubMed Central

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

2007-01-01

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

Altered gene expression in conjunctival squamous cell carcinoma.

PubMed

Mahale, Alka; Alkatan, Hind; Alwadani, Saeed; Othman, Maha; Suarez, Maria J; Price, Antoinette; Al-Hussain, Hailah; Jastaneiah, Sabah; Yu, Wayne; Maktabi, Azza; Deepak, Edward P; Eberhart, Charles G; Asnaghi, Laura

2016-05-01

Conjunctival squamous cell carcinoma is a malignancy of the ocular surface. The molecular drivers responsible for the development and progression of this disease are not well understood. We therefore compared the transcriptional profiles of eight snap-frozen conjunctival squamous cell carcinomas and one in situ lesion with normal conjunctival specimens in order to identify diagnostic markers or therapeutic targets. RNA was analyzed using oligonucleotide microarrays, and a wide range of transcripts with altered expression identified, including many dysregulated in carcinomas arising at other sites. Among the upregulated genes, we observed more than 30-fold induction of the matrix metalloproteinases, MMP-9 and MMP-11, as well as a prominent increase in the mRNA level of a calcium-binding protein important for the intracellular calcium signaling, S100A2, which was induced over 20-fold in the tumor cohort. Clusterin was the most downregulated gene, with an approximately 180-fold reduction in the mRNA expression. These alterations were all confirmed by qPCR in the samples used for initial microarray analysis. In addition, immunohistochemical analysis confirmed the overexpression of MMP-11 and S100A2, as well as reductions in clusterin, in several independent in situ carcinomas of conjunctiva. These data identify a number of alterations, including upregulation of MMP-9, MMP-11, and S100A2, as well as downregulation of clusterin, associated with epithelial tumorigenesis in the ocular surface.

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

PubMed

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

2005-05-18

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

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

Farahani, Poupak; Chiu, Sally; Bowlus, Christopher L.

Obesity is a complex disease. To date, over 100 chromosomal loci for body weight, body fat, regional white adipose tissue weight, and other obesity-related traits have been identified in humans and in animal models. For most loci, the underlying genes are not yet identified; some of these chromosomal loci will be alleles of known obesity genes, whereas many will represent alleles of unknown genes. Microarray analysis allows simultaneous multiple gene and pathway discovery. cDNA and oligonucleotide arrays are commonly used to identify differentially expressed genes by surveys of large numbers of known and unnamed genes. Two papers previously identified genesmore » differentially expressed in adipose tissue of mouse models of obesity and diabetes by analysis of hybridization to Affymetrix oligonucleotide chips.« less

Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

PubMed

Schneider, Uffe Vest

2012-01-01

This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA molecules and other nucleic acid stabilizing molecules can increase analytical sensitivity whilst maintaining nucleobase mismatch discrimination in triplex helix based diagnostic assays.

Data-adaptive test statistics for microarray data.

PubMed

Mukherjee, Sach; Roberts, Stephen J; van der Laan, Mark J

2005-09-01

An important task in microarray data analysis is the selection of genes that are differentially expressed between different tissue samples, such as healthy and diseased. However, microarray data contain an enormous number of dimensions (genes) and very few samples (arrays), a mismatch which poses fundamental statistical problems for the selection process that have defied easy resolution. In this paper, we present a novel approach to the selection of differentially expressed genes in which test statistics are learned from data using a simple notion of reproducibility in selection results as the learning criterion. Reproducibility, as we define it, can be computed without any knowledge of the 'ground-truth', but takes advantage of certain properties of microarray data to provide an asymptotically valid guide to expected loss under the true data-generating distribution. We are therefore able to indirectly minimize expected loss, and obtain results substantially more robust than conventional methods. We apply our method to simulated and oligonucleotide array data. By request to the corresponding author.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Construction of a robust microarray from a non-model species (largemouth bass) using pyrosequencing technology

PubMed Central

Garcia-Reyero, Natàlia; Griffitt, Robert J.; Liu, Li; Kroll, Kevin J.; Farmerie, William G.; Barber, David S.; Denslow, Nancy D.

2009-01-01

A novel custom microarray for largemouth bass (Micropterus salmoides) was designed with sequences obtained from a normalized cDNA library using the 454 Life Sciences GS-20 pyrosequencer. This approach yielded in excess of 58 million bases of high-quality sequence. The sequence information was combined with 2,616 reads obtained by traditional suppressive subtractive hybridizations to derive a total of 31,391 unique sequences. Annotation and coding sequences were predicted for these transcripts where possible. 16,350 annotated transcripts were selected as target sequences for the design of the custom largemouth bass oligonucleotide microarray. The microarray was validated by examining the transcriptomic response in male largemouth bass exposed to 17β-œstradiol. Transcriptomic responses were assessed in liver and gonad, and indicated gene expression profiles typical of exposure to œstradiol. The results demonstrate the potential to rapidly create the tools necessary to assess large scale transcriptional responses in non-model species, paving the way for expanded impact of toxicogenomics in ecotoxicology. PMID:19936325

Differential Transcriptional Response to Nonassociative and Associative Components of Classical Fear Conditioning in the Amygdala and Hippocampus

ERIC Educational Resources Information Center

Isiegas, Carolina; Stein, Joel; Hellman, Kevin; Hannenhalli, Sridhar; Abel, Ted; Keeley, Michael B.; Wood, Marcelo A.

2006-01-01

Classical fear conditioning requires the recognition of conditioned stimuli (CS) and the association of the CS with an aversive stimulus. We used Affymetrix oligonucleotide microarrays to characterize changes in gene expression compared to naive mice in both the amygdala and the hippocampus 30 min after classical fear conditioning and 30 min after…

OY-TES-1 may regulate the malignant behavior of liver cancer via NANOG, CD9, CCND2 and CDCA3: a bioinformatic analysis combine with RNAi and oligonucleotide microarray.

PubMed

Hu, Qiping; Fu, Jun; Luo, Bin; Huang, Miao; Guo, Wenwen; Lin, Yongda; Xie, Xiaoxun; Xiao, Shaowen

2015-04-01

Given its tumor-specific expression, including liver cancer, OY-TES-1 is a potential molecular marker for the diagnosis and immunotherapy of liver cancers. However, investigations of the mechanisms and the role of OY-TES-1 in liver cancer are rare. In the present study, based on a comprehensive bioinformatic analysis combined with RNA interference (RNAi) and oligonucleotide microarray, we report for the first time that downregulation of OY-TES-1 resulted in significant changes in expression of NANOG, CD9, CCND2 and CDCA3 in the liver cancer cell line BEL-7404. NANOG, CD9, CCND2 and CDCA3 may be involved in cell proliferation, migration, invasion and apoptosis, yet also may be functionally related to each other and OY-TES-1. Among these molecules, we identified that NANOG, containing a Kazal-2 binding motif and homeobox, may be the most likely candidate protein interacting with OY-TES-1 in liver cancer. Thus, the present study may provide important information for further investigation of the roles of OY-TES-1 in liver cancer.

Plant-pathogen interactions: what microarray tells about it?

PubMed

Lodha, T D; Basak, J

2012-01-01

Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant-pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes

PubMed Central

Vasson, Aurélie; Leroux, Céline; Orhant, Lucie; Boimard, Mathieu; Toussaint, Aurélie; Leroy, Chrystel; Commere, Virginie; Ghiotti, Tiffany; Deburgrave, Nathalie; Saillour, Yoann; Atlan, Isabelle; Fouveaut, Corinne; Beldjord, Cherif; Valleix, Sophie; Leturcq, France; Dodé, Catherine; Bienvenu, Thierry; Chelly, Jamel; Cossée, Mireille

2013-01-01

The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability ‘homemade' make it a valuable tool as a new diagnostic approach of CNMs. PMID:23340513

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

PubMed Central

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

2006-01-01

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

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

PubMed Central

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

2008-01-01

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

Strategies for comparing gene expression profiles from different microarray platforms: application to a case-control experiment.

PubMed

Severgnini, Marco; Bicciato, Silvio; Mangano, Eleonora; Scarlatti, Francesca; Mezzelani, Alessandra; Mattioli, Michela; Ghidoni, Riccardo; Peano, Clelia; Bonnal, Raoul; Viti, Federica; Milanesi, Luciano; De Bellis, Gianluca; Battaglia, Cristina

2006-06-01

Meta-analysis of microarray data is increasingly important, considering both the availability of multiple platforms using disparate technologies and the accumulation in public repositories of data sets from different laboratories. We addressed the issue of comparing gene expression profiles from two microarray platforms by devising a standardized investigative strategy. We tested this procedure by studying MDA-MB-231 cells, which undergo apoptosis on treatment with resveratrol. Gene expression profiles were obtained using high-density, short-oligonucleotide, single-color microarray platforms: GeneChip (Affymetrix) and CodeLink (Amersham). Interplatform analyses were carried out on 8414 common transcripts represented on both platforms, as identified by LocusLink ID, representing 70.8% and 88.6% of annotated GeneChip and CodeLink features, respectively. We identified 105 differentially expressed genes (DEGs) on CodeLink and 42 DEGs on GeneChip. Among them, only 9 DEGs were commonly identified by both platforms. Multiple analyses (BLAST alignment of probes with target sequences, gene ontology, literature mining, and quantitative real-time PCR) permitted us to investigate the factors contributing to the generation of platform-dependent results in single-color microarray experiments. An effective approach to cross-platform comparison involves microarrays of similar technologies, samples prepared by identical methods, and a standardized battery of bioinformatic and statistical analyses.

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

PubMed

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

2010-04-01

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

Global gene expression profiles of Phytophthora ramorum strain pr102 in response to plant host and tissue differentiation

Treesearch

Caroline M. Press; Niklaus J. Grunwald

2008-01-01

The release of the draft genome sequence of P. ramorum strain Pr102, enabled the construction of an oligonucleotide microarray of the entire genome of Pr102. The array contains 344,680 features (oligos) that represent the transcriptome of Pr102. P. ramorum RNA was extracted from mycelium and sporangia and used to compare gene...

Time-series oligonucleotide count to assign antiviral siRNAs with long utility fit in the big data era.

PubMed

Wada, K; Wada, Y; Iwasaki, Y; Ikemura, T

2017-10-01

Oligonucleotides are key elements of nucleic acid therapeutics such as small interfering RNAs (siRNAs). Influenza and Ebolaviruses are zoonotic RNA viruses mutating very rapidly, and their sequence changes must be characterized intensively to design therapeutic oligonucleotides with long utility. Focusing on a total of 182 experimentally validated siRNAs for influenza A, B and Ebolaviruses compiled by the siRNA database, we conducted time-series analyses of occurrences of siRNA targets in these viral genomes. Reflecting their high mutation rates, occurrences of target oligonucleotides evidently fluctuate in viral populations and often disappear. Time-series analysis of the one-base changed sequences derived from each original target identified the oligonucleotide that shows a compensatory increase and will potentially become the 'awaiting-type oligonucleotide'; the combined use of this oligonucleotide with the original can provide therapeutics with long utility. This strategy is also useful for assigning diagnostic reverse transcription-PCR primers with long utility.

DNA microarrays for identifying fishes.

PubMed

Kochzius, M; Nölte, M; Weber, H; Silkenbeumer, N; Hjörleifsdottir, S; Hreggvidsson, G O; Marteinsson, V; Kappel, K; Planes, S; Tinti, F; Magoulas, A; Garcia Vazquez, E; Turan, C; Hervet, C; Campo Falgueras, D; Antoniou, A; Landi, M; Blohm, D

2008-01-01

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a "Fish Chip" for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

Design and verification of a pangenome microarray oligonucleotide probe set for Dehalococcoides spp.

PubMed

Hug, Laura A; Salehi, Maryam; Nuin, Paulo; Tillier, Elisabeth R; Edwards, Elizabeth A

2011-08-01

Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an oligonucleotide microarray probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This "pangenome" probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome probe set was compared to probe sets designed independently for each of the five Dehalococcoides strains. The pangenome probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific probe sets on nontarget strains with <99% average nucleotide identity. An in silico analysis of the expected probe hybridization against the recently released Dehalococcoides strain GT genome and additional KB-1 metagenome sequence data indicated that the pangenome probe set performs more robustly than the combined strain-specific probe sets in the detection of genes not included in the original design. The pangenome probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

PubMed

Samolski, Ilanit; de Luis, Alberto; Vizcaíno, Juan Antonio; Monte, Enrique; Suárez, M Belén

2009-10-13

It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray

PubMed Central

2009-01-01

Background It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Results Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. Conclusion The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction. PMID:19825185

Emerging Use of Gene Expression Microarrays in Plant Physiology

DOE PAGES

Wullschleger, Stan D.; Difazio, Stephen P.

2003-01-01

Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology weremore » selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.« less

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

PubMed

Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

2016-01-01

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

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

NASA Astrophysics Data System (ADS)

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

2013-10-01

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

Molecular Biology and Prevention of Endometrial Cancer

DTIC Science & Technology

2009-07-01

us time to complete the study. Aim 2: To analyze vaginal and cervical adenocarcinomas , that have arisen in women exposed to DES in- utero , for...therapy. Methods: 1) Oligonucleotide microarray analysis was performed on a panel of endometrial cancers. 2) A subset of adenocarcinoma cases...from the International DES Registry (IDESR) was analyzed for MSI 3) A case-control study of the CASH database was performed to evaluate the

GermOnline 4.0 is a genomics gateway for germline development, meiosis and the mitotic cell cycle.

PubMed

Lardenois, Aurélie; Gattiker, Alexandre; Collin, Olivier; Chalmel, Frédéric; Primig, Michael

2010-01-01

GermOnline 4.0 is a cross-species database portal focusing on high-throughput expression data relevant for germline development, the meiotic cell cycle and mitosis in healthy versus malignant cells. It is thus a source of information for life scientists as well as clinicians who are interested in gene expression and regulatory networks. The GermOnline gateway provides unlimited access to information produced with high-density oligonucleotide microarrays (3'-UTR GeneChips), genome-wide protein-DNA binding assays and protein-protein interaction studies in the context of Ensembl genome annotation. Samples used to produce high-throughput expression data and to carry out genome-wide in vivo DNA binding assays are annotated via the MIAME-compliant Multiomics Information Management and Annotation System (MIMAS 3.0). Furthermore, the Saccharomyces Genomics Viewer (SGV) was developed and integrated into the gateway. SGV is a visualization tool that outputs genome annotation and DNA-strand specific expression data produced with high-density oligonucleotide tiling microarrays (Sc_tlg GeneChips) which cover the complete budding yeast genome on both DNA strands. It facilitates the interpretation of expression levels and transcript structures determined for various cell types cultured under different growth and differentiation conditions. Database URL: www.germonline.org/

GermOnline 4.0 is a genomics gateway for germline development, meiosis and the mitotic cell cycle

PubMed Central

Lardenois, Aurélie; Gattiker, Alexandre; Collin, Olivier; Chalmel, Frédéric; Primig, Michael

2010-01-01

GermOnline 4.0 is a cross-species database portal focusing on high-throughput expression data relevant for germline development, the meiotic cell cycle and mitosis in healthy versus malignant cells. It is thus a source of information for life scientists as well as clinicians who are interested in gene expression and regulatory networks. The GermOnline gateway provides unlimited access to information produced with high-density oligonucleotide microarrays (3′-UTR GeneChips), genome-wide protein–DNA binding assays and protein–protein interaction studies in the context of Ensembl genome annotation. Samples used to produce high-throughput expression data and to carry out genome-wide in vivo DNA binding assays are annotated via the MIAME-compliant Multiomics Information Management and Annotation System (MIMAS 3.0). Furthermore, the Saccharomyces Genomics Viewer (SGV) was developed and integrated into the gateway. SGV is a visualization tool that outputs genome annotation and DNA-strand specific expression data produced with high-density oligonucleotide tiling microarrays (Sc_tlg GeneChips) which cover the complete budding yeast genome on both DNA strands. It facilitates the interpretation of expression levels and transcript structures determined for various cell types cultured under different growth and differentiation conditions. Database URL: www.germonline.org/ PMID:21149299

Multiclass cancer diagnosis using tumor gene expression signatures

DOE PAGES

Ramaswamy, S.; Tamayo, P.; Rifkin, R.; ...

2001-12-11

The optimal treatment of patients with cancer depends on establishing accurate diagnoses by using a complex combination of clinical and histopathological data. In some instances, this task is difficult or impossible because of atypical clinical presentation or histopathology. To determine whether the diagnosis of multiple common adult malignancies could be achieved purely by molecular classification, we subjected 218 tumor samples, spanning 14 common tumor types, and 90 normal tissue samples to oligonucleotide microarray gene expression analysis. The expression levels of 16,063 genes and expressed sequence tags were used to evaluate the accuracy of a multiclass classifier based on a supportmore » vector machine algorithm. Overall classification accuracy was 78%, far exceeding the accuracy of random classification (9%). Poorly differentiated cancers resulted in low-confidence predictions and could not be accurately classified according to their tissue of origin, indicating that they are molecularly distinct entities with dramatically different gene expression patterns compared with their well differentiated counterparts. Taken together, these results demonstrate the feasibility of accurate, multiclass molecular cancer classification and suggest a strategy for future clinical implementation of molecular cancer diagnostics.« less

Using transcriptional profiling to develop a diagnostic test of operational tolerance in liver transplant recipients.

PubMed

Martínez-Llordella, Marc; Lozano, Juan José; Puig-Pey, Isabel; Orlando, Giuseppe; Tisone, Giuseppe; Lerut, Jan; Benítez, Carlos; Pons, Jose Antonio; Parrilla, Pascual; Ramírez, Pablo; Bruguera, Miquel; Rimola, Antoni; Sánchez-Fueyo, Alberto

2008-08-01

A fraction of liver transplant recipients are able to discontinue all immunosuppressive therapies without rejecting their grafts and are said to be operationally tolerant to the transplant. However, accurate identification of these recipients remains a challenge. To design a clinically applicable molecular test of operational tolerance in liver transplantation, we studied transcriptional patterns in the peripheral blood of 80 liver transplant recipients and 16 nontransplanted healthy individuals by employing oligonucleotide microarrays and quantitative real-time PCR. This resulted in the discovery and validation of several gene signatures comprising a modest number of genes capable of identifying tolerant and nontolerant recipients with high accuracy. Multiple peripheral blood lymphocyte subsets contributed to the tolerance-associated transcriptional patterns, although NK and gammadeltaTCR+ T cells exerted the predominant influence. These data suggest that transcriptional profiling of peripheral blood can be employed to identify liver transplant recipients who can discontinue immunosuppressive therapy and that innate immune cells are likely to play a major role in the maintenance of operational tolerance in liver transplantation.

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

PubMed

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

2008-11-01

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

Oligonucleotide DNA microarray profiling of lung adenocarcinoma revealed significant downregulation and deletions of vasoactive intestinal peptide receptor 1.

PubMed

Mlakar, Vid; Strazisar, Mojca; Sok, Mihael; Glavac, Damjan

2010-06-01

The purpose of this study was to find novel gene(s) involved in the development of lung adenocarcinoma (AD). Using DNA microarrays, we identified 31 up-regulated and 8 downregulated genes in 12 AD. Real time PCR was used to measure expression of VIPR1 and SPP1 mRNA and possible losses or gains of genes in 32 AD. We describe significant upregulation of the SPP1 gene, downregulation of VIPR1, and losses of the VIPR1 gene. Our findings complement a proposed VIPR1 tumor suppressor role, in which deletions in the 3p22 chromosome region are an important mechanism leading to loss of the VIPR1 gene.

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

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,…

Time-series oligonucleotide count to assign antiviral siRNAs with long utility fit in the big data era

PubMed Central

Wada, K; Wada, Y; Iwasaki, Y; Ikemura, T

2017-01-01

Oligonucleotides are key elements of nucleic acid therapeutics such as small interfering RNAs (siRNAs). Influenza and Ebolaviruses are zoonotic RNA viruses mutating very rapidly, and their sequence changes must be characterized intensively to design therapeutic oligonucleotides with long utility. Focusing on a total of 182 experimentally validated siRNAs for influenza A, B and Ebolaviruses compiled by the siRNA database, we conducted time-series analyses of occurrences of siRNA targets in these viral genomes. Reflecting their high mutation rates, occurrences of target oligonucleotides evidently fluctuate in viral populations and often disappear. Time-series analysis of the one-base changed sequences derived from each original target identified the oligonucleotide that shows a compensatory increase and will potentially become the ‘awaiting-type oligonucleotide’ the combined use of this oligonucleotide with the original can provide therapeutics with long utility. This strategy is also useful for assigning diagnostic reverse transcription-PCR primers with long utility. PMID:28905886

Microarray labeling extension values: laboratory signatures for Affymetrix GeneChips

PubMed Central

Lee, Yun-Shien; Chen, Chun-Houh; Tsai, Chi-Neu; Tsai, Chia-Lung; Chao, Angel; Wang, Tzu-Hao

2009-01-01

Interlaboratory comparison of microarray data, even when using the same platform, imposes several challenges to scientists. RNA quality, RNA labeling efficiency, hybridization procedures and data-mining tools can all contribute variations in each laboratory. In Affymetrix GeneChips, about 11–20 different 25-mer oligonucleotides are used to measure the level of each transcript. Here, we report that ‘labeling extension values (LEVs)’, which are correlation coefficients between probe intensities and probe positions, are highly correlated with the gene expression levels (GEVs) on eukayotic Affymetrix microarray data. By analyzing LEVs and GEVs in the publicly available 2414 cel files of 20 Affymetrix microarray types covering 13 species, we found that correlations between LEVs and GEVs only exist in eukaryotic RNAs, but not in prokaryotic ones. Surprisingly, Affymetrix results of the same specimens that were analyzed in different laboratories could be clearly differentiated only by LEVs, leading to the identification of ‘laboratory signatures’. In the examined dataset, GSE10797, filtering out high-LEV genes did not compromise the discovery of biological processes that are constructed by differentially expressed genes. In conclusion, LEVs provide a new filtering parameter for microarray analysis of gene expression and it may improve the inter- and intralaboratory comparability of Affymetrix GeneChips data. PMID:19295132

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

PubMed Central

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

2006-01-01

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

Molecular classification of benign prostatic hyperplasia: A gene expression profiling study in a rat model.

PubMed

Hata, Junya; Satoh, Yuichi; Akaihata, Hidenori; Hiraki, Hiroyuki; Ogawa, Soichiro; Haga, Nobuhiro; Ishibashi, Kei; Aikawa, Ken; Kojima, Yoshiyuki

2016-07-01

To characterize the molecular features of benign prostatic hyperplasia by carrying out a gene expression profiling analysis in a rat model. Fetal urogenital sinus isolated from 20-day-old male rat embryo was implanted into a pubertal male rat ventral prostate. The implanted urogenital sinus grew time-dependently, and the pathological findings at 3 weeks after implantation showed epithelial hyperplasia as well as stromal hyperplasia. Whole-genome oligonucleotide microarray analysis utilizing approximately 30 000 oligonucleotide probes was carried out using prostate specimens during the prostate growth process (3 weeks after implantation). Microarray analyses showed 926 upregulated (>2-fold change, P < 0.01) and 3217 downregulated genes (<0.5-fold change, P < 0.01) in benign prostatic hyperplasia specimens compared with normal prostate. Gene ontology analyses of upregulated genes showed predominant genetic themes of involvement in development (162 genes, P = 2.01 × 10(-4) ), response to stimulus (163 genes, P = 7.37 × 10(-13) ) and growth (32 genes, P = 1.93 × 10(-5) ). When we used both normal prostate and non-transplanted urogenital sinuses as controls to identify benign prostatic hyperplasia-specific genes, 507 and 406 genes were upregulated and downregulated, respectively. Functional network and pathway analyses showed that genes associated with apoptosis modulation by heat shock protein 70, interleukin-1, interleukin-2 and interleukin-5 signaling pathways, KIT signaling pathway, and secretin-like G-protein-coupled receptors, class B, were relatively activated during the growth process in the benign prostatic hyperplasia specimens. In contrast, genes associated with cholesterol biosynthesis were relatively inactivated. Our microarray analyses of the benign prostatic hyperplasia model rat might aid in clarifying the molecular mechanism of benign prostatic hyperplasia progression, and identifying molecular targets for benign prostatic hyperplasia treatment. © 2016 The Japanese Urological Association.

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

PubMed Central

2010-01-01

Background As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Methods Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Results Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. Conclusions In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment. PMID:20409345

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray.

PubMed

Xie, Shuwu; Zhu, Yan; Ma, Li; Lu, Yingying; Zhou, Jieyun; Gui, Youlun; Cao, Lin

2010-04-22

As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment.

BioconductorBuntu: a Linux distribution that implements a web-based DNA microarray analysis server.

PubMed

Geeleher, Paul; Morris, Dermot; Hinde, John P; Golden, Aaron

2009-06-01

BioconductorBuntu is a custom distribution of Ubuntu Linux that automatically installs a server-side microarray processing environment, providing a user-friendly web-based GUI to many of the tools developed by the Bioconductor Project, accessible locally or across a network. System installation is via booting off a CD image or by using a Debian package provided to upgrade an existing Ubuntu installation. In its current version, several microarray analysis pipelines are supported including oligonucleotide, dual-or single-dye experiments, including post-processing with Gene Set Enrichment Analysis. BioconductorBuntu is designed to be extensible, by server-side integration of further relevant Bioconductor modules as required, facilitated by its straightforward underlying Python-based infrastructure. BioconductorBuntu offers an ideal environment for the development of processing procedures to facilitate the analysis of next-generation sequencing datasets. BioconductorBuntu is available for download under a creative commons license along with additional documentation and a tutorial from (http://bioinf.nuigalway.ie).

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

PubMed

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

2016-03-01

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

High density DNA loading on the M13 bacteriophage provides access to colorimetric and fluorescent protein microarray biosensors.

PubMed

Domaille, Dylan W; Lee, Ju Hun; Cha, Jennifer N

2013-02-28

We report the synthesis, characterization, and protein sensing capabilities of M13 bacteriophage-DNA bioconjugates. DNA oligonucleotides were conjugated to M13 through acyl hydrazone linkages. In one case, DNAzymes retained their catalytic ability when anchored to the virus coat, and in a separate study, the dynamic nature of the hydrazone allowed for liberation of DNA from the phage under mild conditions.

Gene Expression Profiling of Benign and Malignant Pheochromocytoma

PubMed Central

BROUWERS, FREDERIEKE M.; ELKAHLOUN, ABDEL G.; MUNSON, PETER J.; EISENHOFER, GRAEME; BARB, JENNIFER; LINEHAN, W. MARSTON; LENDERS, JACQUES W.M.; DE KRIJGER, RONALD; MANNELLI, MASSIMO; UDELSMAN, ROBERT; OCAL, IDRIS T.; SHULKIN, BARRY L.; BORNSTEIN, STEFAN R.; BREZA, JAN; KSINANTOVA, LUCIA; PACAK, KAREL

2016-01-01

There are currently no reliable diagnostic and prognostic markers or effective treatments for malignant pheochromocytoma. This study used oligonucleotide microarrays to examine gene expression profiles in pheochromocytomas from 90 patients, including 20 with malignant tumors, the latter including metastases and primary tumors from which metastases developed. Other subgroups of tumors included those defined by tissue norepinephrine compared to epinephrine contents (i.e., noradrenergic versus adrenergic phenotypes), adrenal versus extra-adrenal locations, and presence of germline mutations of genes pre-disposing to the tumor. Correcting for the confounding influence of nora-drenergic versus adrenergic catecholamine phenotype by the analysis of variance revealed a larger and more accurate number of genes that discriminated benign from malignant pheochromocytomas than when the confounding influence of catecholamine phenotype was not considered. Seventy percent of these genes were underexpressed in malignant compared to benign tumors. Similarly, 89% of genes were underexpressed in malignant primary tumors compared to benign tumors, suggesting that malignant potential is largely characterized by a less-differentiated pattern of gene expression. The present database of differentially expressed genes provides a unique resource for mapping the pathways leading to malignancy and for establishing new targets for treatment and diagnostic and prognostic markers of malignant disease. The database may also be useful for examining mechanisms of tumorigenesis and genotype–phenotype relationships. Further progress on the basis of this database can be made from follow-up confirmatory studies, application of bioinformatics approaches for data mining and pathway analyses, testing in pheochromocytoma cell culture and animal model systems, and retrospective and prospective studies of diagnostic markers. PMID:17102123

High-resolution chromosomal microarrays in prenatal diagnosis significantly increase diagnostic power.

PubMed

Oneda, Beatrice; Baldinger, Rosa; Reissmann, Regina; Reshetnikova, Irina; Krejci, Pavel; Masood, Rahim; Ochsenbein-Kölble, Nicole; Bartholdi, Deborah; Steindl, Katharina; Morotti, Denise; Faranda, Marzia; Baumer, Alessandra; Asadollahi, Reza; Joset, Pascal; Niedrist, Dunja; Breymann, Christian; Hebisch, Gundula; Hüsler, Margaret; Mueller, René; Prentl, Elke; Wisser, Josef; Zimmermann, Roland; Rauch, Anita

2014-06-01

The objective of this study was to determine for the first time the reliability and the diagnostic power of high-resolution microarray testing in routine prenatal diagnostics. We applied high-resolution chromosomal microarray testing in 464 cytogenetically normal prenatal samples with any indication for invasive testing. High-resolution testing revealed a diagnostic yield of 6.9% and 1.6% in cases of fetal ultrasound anomalies and cases of advanced maternal age (AMA), respectively, which is similar to previous studies using low-resolution microarrays. In three (0.6%) additional cases with an indication of AMA, an aberration in susceptibility risk loci was detected. Moreover, one case (0.2%) showed an X-linked aberration in a female fetus, a finding relevant for future family planning. We found the rate of cases, in which the parents had to be tested for interpretation of unreported copy number variants (3.7%), and the rate of remaining variants of unknown significance (0.4%) acceptably low. Of note, these findings did not cause termination of pregnancy after expert genetic counseling. The 0.4% rate of confined placental mosaicism was similar to that observed by conventional karyotyping and notably involved a case of placental microdeletion. High-resolution prenatal microarray testing is a reliable technique that increases diagnostic yield by at least 17.3% when compared with conventional karyotyping, without an increase in the frequency of variants of uncertain significance. © 2014 John Wiley & Sons, Ltd.

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Targeted self-assembly of functionalized carbon nanotubes on tumors

DOEpatents

Scheinberg, David A.; McDevitt, Michael R.; Villa, Carlos H.; Mulvey, J. Justin

2018-05-22

Provided herein are methods for delivering a molecule in situ to a cell and for treating a cancer via the in situ delivery. The methods comprise contacting or administering to the cell, as two separate components, a morpholino oligonucleotide comprising a targeting moiety followed by a single wall nanotube construct comprising second morpholino oligonucleotides complementary to the first morpholino oligonucleotides and one or both of a therapeutic or diagnostic payload molecule linked to the single wall nanotube construct. Upon self-assembly of a single wall nanotube complex via hybridization of the first morpholino and second complementary morpholino oligonucleotides at the cell, the payload molecule is delivered. Also provided is the two component self-assembly single wall nanotube system and the single wall nanotube construct comprising the second component.

Transcriptome analysis of salinity stress responses in common wheat using a 22k oligo-DNA microarray.

PubMed

Kawaura, Kanako; Mochida, Keiichi; Yamazaki, Yukiko; Ogihara, Yasunari

2006-04-01

In this study, we constructed a 22k wheat oligo-DNA microarray. A total of 148,676 expressed sequence tags of common wheat were collected from the database of the Wheat Genomics Consortium of Japan. These were grouped into 34,064 contigs, which were then used to design an oligonucleotide DNA microarray. Following a multistep selection of the sense strand, 21,939 60-mer oligo-DNA probes were selected for attachment on the microarray slide. This 22k oligo-DNA microarray was used to examine the transcriptional response of wheat to salt stress. More than 95% of the probes gave reproducible hybridization signals when targeted with RNAs extracted from salt-treated wheat shoots and roots. With the microarray, we identified 1,811 genes whose expressions changed more than 2-fold in response to salt. These included genes known to mediate response to salt, as well as unknown genes, and they were classified into 12 major groups by hierarchical clustering. These gene expression patterns were also confirmed by real-time reverse transcription-PCR. Many of the genes with unknown function were clustered together with genes known to be involved in response to salt stress. Thus, analysis of gene expression patterns combined with gene ontology should help identify the function of the unknown genes. Also, functional analysis of these wheat genes should provide new insight into the response to salt stress. Finally, these results indicate that the 22k oligo-DNA microarray is a reliable method for monitoring global gene expression patterns in wheat.

Ultrasensitive Label-free Electronic Chip for DNA Analysis Using Carbon Nanotube Nanoelectrode Arrays

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Ye, Qi; Han, Jie; Meyyappan, M.

2004-01-01

There is a strong need for faster, cheaper, and simpler methods for nucleic acid analysis in today s clinical tests. Nanotechnologies can potentially provide solutions to these requirements by integrating nanomaterials with biofunctionalities. Dramatic improvement in the sensitivity and multiplexing can be achieved through the high-degree miniaturization. Here, we present our study in the development of an ultrasensitive label-free electronic chip for DNA/RNA analysis based on carbon nanotube nanoelectrode arrays. A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in a SiO2 matrix is fabricated using a bottom-up approach. Characteristic nanoelectrode behavior is observed with a low-density MWNT nanoelectrode array in measuring both the bulk and surface immobilized redox species. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes, with a wide potential window, flexible chemical functionalities, and good biocompatibility. A BRCA1 related oligonucleotide probe with 18 bases is covalently functionalized at the open ends of the MWNTs and specifically hybridized with an oligonucleotide target as well as a PCR amplicon. The guanine bases in the target molecules are employed as the signal moieties for the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. This technique has been employed for direct electrochemical detection of label-free PCR amplicon through specific hybridization with the BRCAl probe. The detection limit is estimated to be less than approximately 1000 DNA molecules, approaching the limit of the sensitivity by laser-based fluorescence techniques in DNA microarray. This system provides a general electronic platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, simple sample preparation, and low- cost operation.

Engineering Improvements in a Bacterial Therapeutic Delivery System for Breast Cancer

DTIC Science & Technology

2009-09-01

curve is observed on the other platform (Supplementary Figure 2H). Although it is not the object of this article to explore a physical explanation for...light-directed oligonucleotide microarrays using a digital micromirror array.. Nat Biotechnol, 17(10), 974–978. [12] Matveeva, O. V., Shabalina, S. A...Rouillard, J. M., Whittam, T. S., Gulari, E., Tiedje, J. M., and Hashsham, S. A. (2006) On- chip non-equilibrium dissociation curves and dissociation

Single-feature polymorphism discovery in the barley transcriptome

PubMed Central

Rostoks, Nils; Borevitz, Justin O; Hedley, Peter E; Russell, Joanne; Mudie, Sharon; Morris, Jenny; Cardle, Linda; Marshall, David F; Waugh, Robbie

2005-01-01

A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes. PMID:15960806

Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer

PubMed Central

Kinoshita, Kenji; Fujimoto, Kentaro; Yakabe, Toru; Saito, Shin; Hamaguchi, Yuzo; Kikuchi, Takayuki; Nonaka, Ken; Murata, Shigenori; Masuda, Daisuke; Takada, Wataru; Funaoka, Sohei; Arai, Susumu; Nakanishi, Hisao; Yokoyama, Kanehisa; Fujiwara, Kazuhiko; Matsubara, Kenichi

2007-01-01

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO® PrimeSurface® with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3′ end of the immobilized DNA primers on the S-Bio® by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA. PMID:17135189

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

PubMed Central

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

2011-01-01

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

Application of Immunosignatures for Diagnosis of Valley Fever

PubMed Central

Navalkar, Krupa Arun; Johnston, Stephen Albert; Woodbury, Neal; Galgiani, John N.; Magee, D. Mitchell; Chicacz, Zbigniew

2014-01-01

Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives. An immunosignature is the pattern of antibody binding to random-sequence peptides on a peptide microarray. A 10,000-peptide microarray was first used to determine whether valley fever patients can be distinguished from 3 other cohorts with similar infections. After determining the VF-specific peptides, a small 96-peptide diagnostic array was created and tested. The performances of the 10,000-peptide array and the 96-peptide diagnostic array were compared to that of the ID diagnostic standard. The 10,000-peptide microarray classified the VF samples from the other 3 infections with 98% accuracy. It also classified VF false-negative patients with 100% sensitivity in a blinded test set versus 28% sensitivity for ID. The immunosignature microarray has potential for simultaneously distinguishing valley fever patients from those with other fungal or bacterial infections. The same 10,000-peptide array can diagnose VF false-negative patients with 100% sensitivity. The smaller 96-peptide diagnostic array was less specific for diagnosing false negatives. We conclude that the performance of the immunosignature diagnostic exceeds that of the existing standard, and the immunosignature can distinguish related infections and might be used in lieu of existing diagnostics. PMID:24964807

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2012-07-01

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

Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa

PubMed Central

Davey, Mark W; Graham, Neil S; Vanholme, Bartel; Swennen, Rony; May, Sean T; Keulemans, Johan

2009-01-01

Background 'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip® microarrays to profile the response of the banana (Musa spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed Musa transcripts. Results Following cross-hybridisation of Musa gDNA to the Rice GeneChip® Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the Musa cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 Musa gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive Musa transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments. Conclusion Our results demonstrate that despite the general paucity of nucleotide sequence data in Musa and only distant phylogenetic relations to rice, gDNA probe-based cross-hybridisation to the Rice GeneChip® is a highly promising strategy to study complex biological responses and illustrates the potential of such strategies for gene discovery in non-model species. PMID:19758430

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

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…

Assessing differential gene expression with small sample sizes in oligonucleotide arrays using a mean-variance model.

PubMed

Hu, Jianhua; Wright, Fred A

2007-03-01

The identification of the genes that are differentially expressed in two-sample microarray experiments remains a difficult problem when the number of arrays is very small. We discuss the implications of using ordinary t-statistics and examine other commonly used variants. For oligonucleotide arrays with multiple probes per gene, we introduce a simple model relating the mean and variance of expression, possibly with gene-specific random effects. Parameter estimates from the model have natural shrinkage properties that guard against inappropriately small variance estimates, and the model is used to obtain a differential expression statistic. A limiting value to the positive false discovery rate (pFDR) for ordinary t-tests provides motivation for our use of the data structure to improve variance estimates. Our approach performs well compared to other proposed approaches in terms of the false discovery rate.

Androgen Metabolism in Progression to Androgen-Independent Prostate Cancer

DTIC Science & Technology

2009-06-01

transcripts (TMPRSS2 exon 2-ERG exon 4) in 11 of 29 cases. Affymetrix oligonucleotide microarray data on these tumors versus a group of 27...medium were treated with DHT and immunoblotted. B, RT-PCR for ERG ( exon 9/10), TMPRSS2 ( exon 5/6), and PSA mRNA after DHT stimulation. C, cells in...therapeutic index CYP3A4 substrates were excluded. The treatment was ketoconazole 400 mg po TID, hydrocortisone (30 mg in AM and 10 mg in PM) and

UniPROBE, update 2011: expanded content and search tools in the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Robasky, Kimberly; Bulyk, Martha L

2011-01-01

The Universal PBM Resource for Oligonucleotide-Binding Evaluation (UniPROBE) database is a centralized repository of information on the DNA-binding preferences of proteins as determined by universal protein-binding microarray (PBM) technology. Each entry for a protein (or protein complex) in UniPROBE provides the quantitative preferences for all possible nucleotide sequence variants ('words') of length k ('k-mers'), as well as position weight matrix (PWM) and graphical sequence logo representations of the k-mer data. In this update, we describe >130% expansion of the database content, incorporation of a protein BLAST (blastp) tool for finding protein sequence matches in UniPROBE, the introduction of UniPROBE accession numbers and additional database enhancements. The UniPROBE database is available at http://uniprobe.org.

DNA Microarray for Detection of Macrolide Resistance Genes

PubMed Central

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

2006-01-01

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

Microarray-based gene expression profiling to elucidate effectiveness of fermented Codonopsis lanceolata in mice.

PubMed

Choi, Woon Yong; Kim, Ji Seon; Park, Sung Jin; Ma, Choong Je; Lee, Hyeon Yong

2014-04-08

In this study, the effect of Codonopsis lanceolata fermented by lactic acid on controlling gene expression levels related to obesity was observed in an oligonucleotide chip microarray. Among 8170 genes, 393 genes were up regulated and 760 genes were down regulated in feeding the fermented C. lanceolata (FCL). Another 374 genes were up regulated and 527 genes down regulated without feeding the sample. The genes were not affected by the FCL sample. It was interesting that among those genes, Chytochrome P450, Dmbt1, LOC76487, and thyroid hormones, etc., were mostly up or down regulated. These genes are more related to lipid synthesis. We could conclude that the FCL possibly controlled the gene expression levels related to lipid synthesis, which resulted in reducing obesity. However, more detailed protein expression experiments should be carried out.

The GermOnline cross-species systems browser provides comprehensive information on genes and gene products relevant for sexual reproduction.

PubMed

Gattiker, Alexandre; Niederhauser-Wiederkehr, Christa; Moore, James; Hermida, Leandro; Primig, Michael

2007-01-01

We report a novel release of the GermOnline knowledgebase covering genes relevant for the cell cycle, gametogenesis and fertility. GermOnline was extended into a cross-species systems browser including information on DNA sequence annotation, gene expression and the function of gene products. The database covers eight model organisms and Homo sapiens, for which complete genome annotation data are available. The database is now built around a sophisticated genome browser (Ensembl), our own microarray information management and annotation system (MIMAS) used to extensively describe experimental data obtained with high-density oligonucleotide microarrays (GeneChips) and a comprehensive system for online editing of database entries (MediaWiki). The RNA data include results from classical microarrays as well as tiling arrays that yield information on RNA expression levels, transcript start sites and lengths as well as exon composition. Members of the research community are solicited to help GermOnline curators keep database entries on genes and gene products complete and accurate. The database is accessible at http://www.germonline.org/.

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

PubMed

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

2013-09-26

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

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

PubMed Central

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

2013-01-01

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

Evaluating concentration estimation errors in ELISA microarray experiments

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

Daly, Don S.; White, Amanda M.; Varnum, Susan M.

Enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay to predict a protein concentration in a sample. Deploying ELISA in a microarray format permits simultaneous prediction of the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Evaluating prediction error is critical to interpreting biological significance and improving the ELISA microarray process. Evaluating prediction error must be automated to realize a reliable high-throughput ELISA microarray system. Methods: In this paper, we present a statistical method based on propagation of error to evaluate prediction errors in the ELISA microarray process. Althoughmore » propagation of error is central to this method, it is effective only when comparable data are available. Therefore, we briefly discuss the roles of experimental design, data screening, normalization and statistical diagnostics when evaluating ELISA microarray prediction errors. We use an ELISA microarray investigation of breast cancer biomarkers to illustrate the evaluation of prediction errors. The illustration begins with a description of the design and resulting data, followed by a brief discussion of data screening and normalization. In our illustration, we fit a standard curve to the screened and normalized data, review the modeling diagnostics, and apply propagation of error.« less

Use of continuous/contiguous stacking hybridization as a diagnostic tool

DOEpatents

Mirzabekov, Andrei Darievich; Yershov, Gennadiy Moseyevich; Kirillov, Eugene Vladislavovich; Parinov, Sergei Valeryevich; Barski, Victor Evgenievich; Lysov, Yuri Petrovich

1999-01-01

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates.

Use of continuous/contiguous stacking hybridization as a diagnostic tool

DOEpatents

Mirzabekov, A.D.; Yershov, G.M.; Kirillov, E.V.; Parinov, S.V.; Barski, V.E.; Lysov, Y.P.

1999-06-01

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates. 5 figs.

Silver Nanoparticle Oligonucleotide Conjugates Based on DNA with Triple Cyclic Disulfide Moieties

PubMed Central

Lee, Jae-Seung; Lytton-Jean, Abigail K. R.; Hurst, Sarah J.; Mirkin, Chad A.

2011-01-01

We report a new strategy for preparing silver nanoparticle oligonucleotide conjugates that are based upon DNA with cyclic disulfide-anchoring groups. These particles are extremely stable and can withstand NaCl concentrations up to 1.0 M. When silver nanoparticles functionalized with complementary sequences are combined, they assemble to form DNA-linked nanoparticle networks. This assembly process is reversible with heating and is associated with a red-shifting of the particle surface plasmon resonance and a concomitant color change from yellow to pale red. Analogous to the oligonucleotide-functionalized gold nanoparticles, these particles also exhibit highly cooperative binding properties with extremely sharp melting transitions. This work is an important step towards being able to use silver nanoparticle oligonucleotide conjugates for a variety of purposes, including molecular diagnostic labels, synthons in programmable materials synthesis approaches, and functional components for nanoelectronic and plasmonic devices. PMID:17571909

DNA microarray technology in nutraceutical and food safety.

PubMed

Liu-Stratton, Yiwen; Roy, Sashwati; Sen, Chandan K

2004-04-15

The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Analytical Protein Microarrays: Advancements Towards Clinical Applications

PubMed Central

Sauer, Ursula

2017-01-01

Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems. PMID:28146048

A cDNA microarray gene expression data classifier for clinical diagnostics based on graph theory.

PubMed

Benso, Alfredo; Di Carlo, Stefano; Politano, Gianfranco

2011-01-01

Despite great advances in discovering cancer molecular profiles, the proper application of microarray technology to routine clinical diagnostics is still a challenge. Current practices in the classification of microarrays' data show two main limitations: the reliability of the training data sets used to build the classifiers, and the classifiers' performances, especially when the sample to be classified does not belong to any of the available classes. In this case, state-of-the-art algorithms usually produce a high rate of false positives that, in real diagnostic applications, are unacceptable. To address this problem, this paper presents a new cDNA microarray data classification algorithm based on graph theory and is able to overcome most of the limitations of known classification methodologies. The classifier works by analyzing gene expression data organized in an innovative data structure based on graphs, where vertices correspond to genes and edges to gene expression relationships. To demonstrate the novelty of the proposed approach, the authors present an experimental performance comparison between the proposed classifier and several state-of-the-art classification algorithms.

Refinement of light-responsive transcript lists using rice oligonucleotide arrays: evaluation of gene-redundancy.

PubMed

Jung, Ki-Hong; Dardick, Christopher; Bartley, Laura E; Cao, Peijian; Phetsom, Jirapa; Canlas, Patrick; Seo, Young-Su; Shultz, Michael; Ouyang, Shu; Yuan, Qiaoping; Frank, Bryan C; Ly, Eugene; Zheng, Li; Jia, Yi; Hsia, An-Ping; An, Kyungsook; Chou, Hui-Hsien; Rocke, David; Lee, Geun Cheol; Schnable, Patrick S; An, Gynheung; Buell, C Robin; Ronald, Pamela C

2008-10-06

Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org). The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics.

Customized oligonucleotide microarray gene expression-based classification of neuroblastoma patients outperforms current clinical risk stratification.

PubMed

Oberthuer, André; Berthold, Frank; Warnat, Patrick; Hero, Barbara; Kahlert, Yvonne; Spitz, Rüdiger; Ernestus, Karen; König, Rainer; Haas, Stefan; Eils, Roland; Schwab, Manfred; Brors, Benedikt; Westermann, Frank; Fischer, Matthias

2006-11-01

To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease. Two hundred fifty-one neuroblastoma specimens were analyzed using a customized oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States. The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival [EFS] 0.86 +/- 0.03 [favorable; n = 115] v 0.52 +/- 0.07 [unfavorable; n = 59] and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P < .0001) and separated risk groups of current neuroblastoma trials into subgroups with divergent outcome (NB2004: low-risk 3-year EFS 0.86 +/- 0.04 v 0.25 +/- 0.15, P < .0001; intermediate-risk 1.00 v 0.57 +/- 0.19, P = .018; high-risk 0.81 +/- 0.10 v 0.56 +/- 0.08, P = .06). In a multivariate Cox regression model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 [95% CI, 2.544 to 8.893]). Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.

Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA ▿

PubMed Central

Javier, David J.; Castellanos-Gonzalez, Alejandro; Weigum, Shannon E.; White, A. Clinton; Richards-Kortum, Rebecca

2009-01-01

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 103 purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection. PMID:19828740

Performance of seven serological assays for diagnosing tularemia

PubMed Central

2014-01-01

Background Tularemia is a rare zoonotic disease caused by the Gram-negative bacterium Francisella tularensis. Serology is frequently the preferred diagnostic approach, because the pathogen is highly infectious and difficult to cultivate. The aim of this retrospective study was to determine the diagnostic accuracy of tularemia specific tests. Methods The Serazym®Anti-Francisella tularensis ELISA, Serion ELISA classic Francisella tularensis IgG/IgM, an in-house ELISA, the VIRapid® Tularemia immunochromatographic test, an in-house antigen microarray, and a Western Blot (WB) assay were evaluated. The diagnosis tularemia was established using a standard micro-agglutination assay. In total, 135 sera from a series of 110 consecutive tularemia patients were tested. Results The diagnostic sensitivity and diagnostic specificity of the tests were VIRapid (97.0% and 84.0%), Serion IgG (96.3% and 96.8%), Serion IgM (94.8% and 96.8%), Serazym (97.0% and 91.5%), in-house ELISA (95.6% and 76.6%), WB (93.3% and 83.0%), microarray (91.1% and 97.9%). Conclusions The diagnostic value of the commercial assays was proven, because the diagnostic accuracy was >90%. The diagnostic sensitivity of the in-house ELISA and the WB were acceptable, but the diagnostic accuracy was <90%. Interestingly, the antigen microarray test was very specific and had a very good positive predictive value. PMID:24885274

Designing oligo libraries taking alternative splicing into account

NASA Astrophysics Data System (ADS)

Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

2001-06-01

We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

SigReannot-mart: a query environment for expression microarray probe re-annotations.

PubMed

Moreews, François; Rauffet, Gaelle; Dehais, Patrice; Klopp, Christophe

2011-01-01

Expression microarrays are commonly used to study transcriptomes. Most of the arrays are now based on oligo-nucleotide probes. Probe design being a tedious task, it often takes place once at the beginning of the project. The oligo set is then used for several years. During this time period, the knowledge gathered by the community on the genome and the transcriptome increases and gets more precise. Therefore re-annotating the set is essential to supply the biologists with up-to-date annotations. SigReannot-mart is a query environment populated with regularly updated annotations for different oligo sets. It stores the results of the SigReannot pipeline that has mainly been used on farm and aquaculture species. It permits easy extraction in different formats using filters. It is used to compare probe sets on different criteria, to choose the set for a given experiment to mix probe sets in order to create a new one.

Biological data warehousing system for identifying transcriptional regulatory sites from gene expressions of microarray data.

PubMed

Tsou, Ann-Ping; Sun, Yi-Ming; Liu, Chia-Lin; Huang, Hsien-Da; Horng, Jorng-Tzong; Tsai, Meng-Feng; Liu, Baw-Juine

2006-07-01

Identification of transcriptional regulatory sites plays an important role in the investigation of gene regulation. For this propose, we designed and implemented a data warehouse to integrate multiple heterogeneous biological data sources with data types such as text-file, XML, image, MySQL database model, and Oracle database model. The utility of the biological data warehouse in predicting transcriptional regulatory sites of coregulated genes was explored using a synexpression group derived from a microarray study. Both of the binding sites of known transcription factors and predicted over-represented (OR) oligonucleotides were demonstrated for the gene group. The potential biological roles of both known nucleotides and one OR nucleotide were demonstrated using bioassays. Therefore, the results from the wet-lab experiments reinforce the power and utility of the data warehouse as an approach to the genome-wide search for important transcription regulatory elements that are the key to many complex biological systems.

Molecular diagnostics using magnetic nanobeads

NASA Astrophysics Data System (ADS)

Zardán Gómez de la Torre, Teresa; Strömberg, Mattias; Göransson, Jenny; Gunnarsson, Klas; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria

2010-01-01

In this paper, we investigate the volume-amplified magnetic nanobead detection assay with respect to bead size, bead concentration and bead oligonucleotide surface coverage in order to improve the understanding of the underlying microscopic mechanisms. It has been shown that: (i) the immobilization efficiency of the beads depends on the surface coverage of oligonucleotides, (ii) by using lower amounts of probe-tagged beads, detection sensitivity can be improved and (iii) using small enough beads enables both turn-off and turn-on detection. Finally, biplex detection was demonstrated.

Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach

NASA Technical Reports Server (NTRS)

Liu, W. T.; Mirzabekov, A. D.; Stahl, D. A.

2001-01-01

The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

PubMed

Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

2004-03-01

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Miniaturized multiplex label-free electronic chip for rapid nucleic acid analysis based on carbon nanotube nanoelectrode arrays

NASA Technical Reports Server (NTRS)

Koehne, Jessica E.; Chen, Hua; Cassell, Alan M.; Ye, Qi; Han, Jie; Meyyappan, Meyya; Li, Jun

2004-01-01

BACKGROUND: Reducing cost and time is the major concern in clinical diagnostics, particularly in molecular diagnostics. Miniaturization technologies have been recognized as promising solutions to provide low-cost microchips for diagnostics. With the recent advancement in nanotechnologies, it is possible to further improve detection sensitivity and simplify sample preparation by incorporating nanoscale elements in diagnostics devices. A fusion of micro- and nanotechnologies with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices. APPROACH: Vertically aligned multiwalled carbon nanotubes (MWNTs) are fabricated on predeposited microelectrode pads and encapsulated in SiO2 dielectrics with only the very end exposed at the surface to form an inlaid nanoelectrode array (NEA). The NEA is used to collect the electrochemical signal associated with the target molecules binding to the probe molecules, which are covalently attached to the end of the MWNTs. CONTENT: A 3 x 3 microelectrode array is presented to demonstrate the miniaturization and multiplexing capability. A randomly distributed MWNT NEA is fabricated on each microelectrode pad. Selective functionalization of the MWNT end with a specific oligonucleotide probe and passivation of the SiO2 surface with ethylene glycol moieties are discussed. Ru(bpy)2+ -mediator-amplified guanine oxidation is used to directly measure the electrochemical signal associated with target molecules. SUMMARY: The discussed MWNT NEAs have ultrahigh sensitivity in direct electrochemical detection of guanine bases in the nucleic acid target. Fewer than approximately 1000 target nucleic acid molecules can be measured with a single microelectrode pad of approximately 20 x 20 microm2, which approaches the detection limit of laser scanners in fluorescence-based DNA microarray techniques. MWNT NEAs can be easily integrated with microelectronic circuitry and microfluidics for development of a fully automated system for rapid molecular analysis with minimum cost.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

PubMed

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

PubMed Central

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence.

PubMed

Drost, Derek R; Novaes, Evandro; Boaventura-Novaes, Carolina; Benedict, Catherine I; Brown, Ryan S; Yin, Tongming; Tuskan, Gerald A; Kirst, Matias

2009-06-01

Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ-synthesized microarray probes. Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa. A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.

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

PubMed

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

2004-12-01

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

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

PubMed Central

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

2004-01-01

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

DISC-BASED IMMUNOASSAY MICROARRAYS. (R825433)

EPA Science Inventory

Microarray technology as applied to areas that include genomics, diagnostics, environmental, and drug discovery, is an interesting research topic for which different chip-based devices have been developed. As an alternative, we have explored the principle of compact disc-based...

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

PubMed Central

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

2011-01-01

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

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

PubMed Central

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

2003-01-01

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

A comprehensive transcript index of the human genome generated using microarrays and computational approaches

PubMed Central

Schadt, Eric E; Edwards, Stephen W; GuhaThakurta, Debraj; Holder, Dan; Ying, Lisa; Svetnik, Vladimir; Leonardson, Amy; Hart, Kyle W; Russell, Archie; Li, Guoya; Cavet, Guy; Castle, John; McDonagh, Paul; Kan, Zhengyan; Chen, Ronghua; Kasarskis, Andrew; Margarint, Mihai; Caceres, Ramon M; Johnson, Jason M; Armour, Christopher D; Garrett-Engele, Philip W; Tsinoremas, Nicholas F; Shoemaker, Daniel D

2004-01-01

Background Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22. Results The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale. Conclusions These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized. PMID:15461792

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

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

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

2000-12-01

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

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

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

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

2001-07-05

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

Gene Expression Profiling of Gastric Cancer

PubMed Central

Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

2015-01-01

Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

An Efficient Covalent Coating on Glass Slides for Preparation of Optical Oligonucleotide Microarrays

PubMed Central

Pourjahed, Atefeh; Rabiee, Mohammad; Tahriri, Mohammadreza

2013-01-01

Objective(s): Microarrays are potential analyzing tools for genomics and proteomics researches, which is in needed of suitable substrate for coating and also hybridization of biomolecules. Materials and Methods: In this research, a thin film of oxidized agarose was prepared on the glass slides which previously coated with poly-L-lysine (PLL). Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups; also bound to the amino groups of biomolecules. These linkages were fixed by UV irradiation. The prepared substrates were compared to only agarose-coated and PLL-coated slides. Results: Results on atomic force microscope (AFM) demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. In addition, the signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively. Conclusion: The agarose-PLL microarrays had the highest signal (2546) and lowest background signal (205) in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes. PMID:24570832

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

PubMed

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

2008-10-01

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

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

PubMed Central

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

2008-01-01

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

High-throughput microarray technology in diagnostics of enterobacteria based on genome-wide probe selection and regression analysis.

PubMed

Friedrich, Torben; Rahmann, Sven; Weigel, Wilfried; Rabsch, Wolfgang; Fruth, Angelika; Ron, Eliora; Gunzer, Florian; Dandekar, Thomas; Hacker, Jörg; Müller, Tobias; Dobrindt, Ulrich

2010-10-21

The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.

Species-Level Identification of Orthopoxviruses with an Oligonucleotide Microchip

PubMed Central

Lapa, Sergey; Mikheev, Maxim; Shchelkunov, Sergei; Mikhailovich, Vladimir; Sobolev, Alexander; Blinov, Vladimir; Babkin, Igor; Guskov, Alexander; Sokunova, Elena; Zasedatelev, Alexander; Sandakhchiev, Lev; Mirzabekov, Andrei

2002-01-01

A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used). PMID:11880388

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-01-01

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

Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization

PubMed Central

Sabath, Daniel E.; Bender, Michael A.; Sankaran, Vijay G.; Vamos, Esther; Kentsis, Alex; Yi, Hye-Son; Greisman, Harvey A.

2017-01-01

Thalassemia is among the most common genetic diseases worldwide. α-Thalassemia is usually caused by deletion of one or more of the duplicated HBA genes on chromosome 16. In contrast, most β-thalassemia results from point mutations that decrease or eliminate expression of the HBB gene on chromosome 11. Deletions within the HBB locus result in thalassemia or hereditary persistence of fetal Hb. Although routine diagnostic testing cannot distinguish thalassemia deletions from point mutations, deletional hereditary persistence of fetal Hb is notable for having an elevated HbF level with a normal mean corpuscular volume. A small number of deletions accounts for most α-thalassemias; in contrast, there are no predominant HBB deletions causing β-thalassemia. To facilitate the identification and characterization of deletions of the HBA and HBB globin loci, we performed array-based comparative genomic hybridization using a custom oligonucleotide microarray. We accurately mapped the breakpoints of known and previously uncharacterized HBB deletions defining previously uncharacterized deletion breakpoints by PCR amplification and sequencing. The array also successfully identified the common HBA deletions --SEA and --FIL. In summary, comparative genomic hybridization can be used to characterize deletions of the HBA and HBB loci, allowing high-resolution characterization of novel deletions that are not readily detected by PCR-based methods. PMID:26612711

Tissue microarrays and quantitative tissue-based image analysis as a tool for oncology biomarker and diagnostic development.

PubMed

Dolled-Filhart, Marisa P; Gustavson, Mark D

2012-11-01

Translational oncology has been improved by using tissue microarrays (TMAs), which facilitate biomarker analysis of large cohorts on a single slide. This has allowed for rapid analysis and validation of potential biomarkers for prognostic and predictive value, as well as for evaluation of biomarker prevalence. Coupled with quantitative analysis of immunohistochemical (IHC) staining, objective and standardized biomarker data from tumor samples can further advance companion diagnostic approaches for the identification of drug-responsive or resistant patient subpopulations. This review covers the advantages, disadvantages and applications of TMAs for biomarker research. Research literature and reviews of TMAs and quantitative image analysis methodology have been surveyed for this review (with an AQUA® analysis focus). Applications such as multi-marker diagnostic development and pathway-based biomarker subpopulation analyses are described. Tissue microarrays are a useful tool for biomarker analyses including prevalence surveys, disease progression assessment and addressing potential prognostic or predictive value. By combining quantitative image analysis with TMAs, analyses will be more objective and reproducible, allowing for more robust IHC-based diagnostic test development. Quantitative multi-biomarker IHC diagnostic tests that can predict drug response will allow for greater success of clinical trials for targeted therapies and provide more personalized clinical decision making.

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

PubMed

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

2018-05-21

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

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

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

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

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

PubMed Central

Kostić, Tanja; Sessitsch, Angela

2011-01-01

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

Chromosomal Microarray Testing in 42 Korean Patients with Unexplained Developmental Delay, Intellectual Disability, Autism Spectrum Disorders, and Multiple Congenital Anomalies.

PubMed

Lee, Sun Ho; Song, Wung Joo

2017-09-01

Chromosomal microarray (CMA) is a high-resolution, high-throughput method of identifying submicroscopic genomic copy number variations (CNVs). CMA has been established as the first-line diagnostic test for individuals with developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), and multiple congenital anomalies (MCAs). CMA analysis was performed in 42 Korean patients who had been diagnosed with unexplained DD, ID, ASDs, and MCAs. Clinically relevant CNVs were discovered in 28 patients. Variants of unknown significance were detected in 13 patients. The diagnostic yield was high (66.7%). CMA is a superior diagnostic tool compared with conventional karyotyping and fluorescent in situ hybridization.

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

White, Amanda M.; Daly, Don S.; Willse, Alan R.

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.

The Diagnostic Yield of Array Comparative Genomic Hybridization Is High Regardless of Severity of Intellectual Disability/Developmental Delay in Children.

PubMed

D'Arrigo, Stefano; Gavazzi, Francesco; Alfei, Enrico; Zuffardi, Orsetta; Montomoli, Cristina; Corso, Barbara; Buzzi, Erika; Sciacca, Francesca L; Bulgheroni, Sara; Riva, Daria; Pantaleoni, Chiara

2016-05-01

Microarray-based comparative genomic hybridization is a method of molecular analysis that identifies chromosomal anomalies (or copy number variants) that correlate with clinical phenotypes. The aim of the present study was to apply a clinical score previously designated by de Vries to 329 patients with intellectual disability/developmental disorder (intellectual disability/developmental delay) referred to our tertiary center and to see whether the clinical factors are associated with a positive outcome of aCGH analyses. Another goal was to test the association between a positive microarray-based comparative genomic hybridization result and the severity of intellectual disability/developmental delay. Microarray-based comparative genomic hybridization identified structural chromosomal alterations responsible for the intellectual disability/developmental delay phenotype in 16% of our sample. Our study showed that causative copy number variants are frequently found even in cases of mild intellectual disability (30.77%). We want to emphasize the need to conduct microarray-based comparative genomic hybridization on all individuals with intellectual disability/developmental delay, regardless of the severity, because the degree of intellectual disability/developmental delay does not predict the diagnostic yield of microarray-based comparative genomic hybridization. © The Author(s) 2015.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

PubMed Central

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

2009-01-01

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

Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

PubMed

Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

2018-04-01

Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and validation of a mixed-tissue oligonucleotide DNA microarray for Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758).

PubMed

Trumbić, Željka; Bekaert, Michaël; Taggart, John B; Bron, James E; Gharbi, Karim; Mladineo, Ivona

2015-11-25

The largest of the tuna species, Atlantic bluefin tuna (Thunnus thynnus), inhabits the North Atlantic Ocean and the Mediterranean Sea and is considered to be an endangered species, largely a consequence of overfishing. T. thynnus aquaculture, referred to as fattening or farming, is a capture based activity dependent on yearly renewal from the wild. Thus, the development of aquaculture practices independent of wild resources can provide an important contribution towards ensuring security and sustainability of this species in the longer-term. The development of such practices is today greatly assisted by large scale transcriptomic studies. We have used pyrosequencing technology to sequence a mixed-tissue normalised cDNA library, derived from adult T. thynnus. A total of 976,904 raw sequence reads were assembled into 33,105 unique transcripts having a mean length of 893 bases and an N50 of 870. Of these, 33.4% showed similarity to known proteins or gene transcripts and 86.6% of them were matched to the congeneric Pacific bluefin tuna (Thunnus orientalis) genome, compared to 70.3% for the more distantly related Nile tilapia (Oreochromis niloticus) genome. Transcript sequences were used to develop a novel 15 K Agilent oligonucleotide DNA microarray for T. thynnus and comparative tissue gene expression profiles were inferred for gill, heart, liver, ovaries and testes. Functional contrasts were strongest between gills and ovaries. Gills were particularly associated with immune system, signal transduction and cell communication, while ovaries displayed signatures of glycan biosynthesis, nucleotide metabolism, transcription, translation, replication and repair. Sequence data generated from a novel mixed-tissue T. thynnus cDNA library provide an important transcriptomic resource that can be further employed for study of various aspects of T. thynnus ecology and genomics, with strong applications in aquaculture. Tissue-specific gene expression profiles inferred through the use of novel oligo-microarray can serve in the design of new and more focused transcriptomic studies for future research of tuna physiology and assessment of the welfare in a production environment.

DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp.

PubMed

Schmoock, Gernot; Ehricht, Ralf; Melzer, Falk; Rassbach, Astrid; Scholz, Holger C; Neubauer, Heinrich; Sachse, Konrad; Mota, Rinaldo Aparecido; Saqib, Muhammad; Elschner, Mandy

2009-01-01

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.

Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

NASA Astrophysics Data System (ADS)

Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

2018-04-01

DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

PubMed

Bidard, Frédérique; Imbeaud, Sandrine; Reymond, Nancie; Lespinet, Olivier; Silar, Philippe; Clavé, Corinne; Delacroix, Hervé; Berteaux-Lecellier, Véronique; Debuchy, Robert

2010-06-18

The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

Use of continuous/contiguous stacking hybridization as a diagnostic tool

DOEpatents

Mirzabekov, Andrei Darievich; Kirillov, Eugene Vladislavovich; Parinov, Sergei Valeryevich; Barski, Victor Evgenievich; Dubiley, Svetlana Alekseevna

2002-01-01

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates. A method is also provided for determining the length of a repeat sequence in DNA or RNA, and also for determining the base sequence of unknown DNA or RNA.

Use of continuous/contiguous stacking hybridization as a diagnostic tool

DOEpatents

Mirzabekov, Andrei Darievich; Kirillov, Eugene Vladislavovich; Parinov, Sergei Valeryevich; Barski, Victor Evgenievich; Dubiley, Svetlana Alekseevna

2000-01-01

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates. A method is also provided for determining the length of a repeat sequence in DNA or RNA, and also for determining the base sequence of unknown DNA or RNA.

Analytical Devices Based on Direct Synthesis of DNA on Paper.

PubMed

Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

2016-01-05

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

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.

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

PubMed

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

2018-03-12

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

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

PubMed Central

Sachse, Konrad; Ziegler, Ute; Keller, Markus

2017-01-01

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

arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays

PubMed Central

Menten, Björn; Pattyn, Filip; De Preter, Katleen; Robbrecht, Piet; Michels, Evi; Buysse, Karen; Mortier, Geert; De Paepe, Anne; van Vooren, Steven; Vermeesch, Joris; Moreau, Yves; De Moor, Bart; Vermeulen, Stefan; Speleman, Frank; Vandesompele, Jo

2005-01-01

Background The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. Results We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. Conclusion ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at . PMID:15910681

Chromosome r(10)(p15.3q26.12) in a newborn child: case report.

PubMed

Gunnarsson, Cecilia; Graffmann, Barbara; Jonasson, Jon

2009-12-07

Ring chromosome 10 is a rare cytogenetic finding. Of the less than 10 reported cases we have found in the literature, none was characterized using high-resolution microarray analysis. Ring chromosomes are frequently unstable due to sister chromatid exchanges and mitotic failures. When mosaicism is present, the interpretation of genotype-phenotype correlations becomes extremely difficult. We report on a newborn girl with growth retardation, microcephaly, congenital heart defects, dysmorphic features and psychomotor retardation. Karyotyping revealed a non-mosaic apparently stable ring chromosome 10 replacing one of the normal homologues in all analyzed metaphases. High-resolution oligonucleotide microarray analysis showed a de novo approximately 12.5 Mb terminal deletion 10q26.12 -> qter and a corresponding 285 kb terminal deletion of 10pter -> p15.3. This case demonstrates that an increased nuchal translucency thickness detected by early ultrasonography should preferably lead to not only QF-PCR for the diagnosis of Down syndrome but also karyotyping. In the future, microarray analysis, which needs further evaluation, might become the method of choice. The clinical phenotype of our patient was in agreement with that of patients with a terminal 10q deletion. For the purpose of genotype-phenotype analysis, there seems to be no need for a "ring syndrome" concept.

Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis

PubMed Central

Wapner, Ronald J.; Martin, Christa Lese; Levy, Brynn; Ballif, Blake C.; Eng, Christine M.; Zachary, Julia M.; Savage, Melissa; Platt, Lawrence D.; Saltzman, Daniel; Grobman, William A.; Klugman, Susan; Scholl, Thomas; Simpson, Joe Leigh; McCall, Kimberly; Aggarwal, Vimla S.; Bunke, Brian; Nahum, Odelia; Patel, Ankita; Lamb, Allen N.; Thom, Elizabeth A.; Beaudet, Arthur L.; Ledbetter, David H.; Shaffer, Lisa G.; Jackson, Laird

2013-01-01

Background Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Methods Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. Results We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down’s syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. Conclusions In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.) PMID:23215555

Gene Expression Changes in Phosphorus Deficient Potato (Solanum tuberosum L.) Leaves and the Potential for Diagnostic Gene Expression Markers

PubMed Central

Hammond, John P.; Broadley, Martin R.; Bowen, Helen C.; Spracklen, William P.; Hayden, Rory M.; White, Philip J.

2011-01-01

Background There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples. Results We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes. Conclusions This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture. PMID:21935429

A functional genomics tool for the Pacific bluefin tuna: Development of a 44K oligonucleotide microarray from whole-genome sequencing data for global transcriptome analysis.

PubMed

Yasuike, Motoshige; Fujiwara, Atushi; Nakamura, Yoji; Iwasaki, Yuki; Nishiki, Issei; Sugaya, Takuma; Shimizu, Akio; Sano, Motohiko; Kobayashi, Takanori; Ototake, Mitsuru

2016-02-01

Bluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis, has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia.

PubMed

Segara, Davendra; Biankin, Andrew V; Kench, James G; Langusch, Catherine C; Dawson, Amanda C; Skalicky, David A; Gotley, David C; Coleman, Maxwell J; Sutherland, Robert L; Henshall, Susan M

2005-05-01

Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARalpha, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and PanIN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early PanIN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses

NASA Technical Reports Server (NTRS)

Urakawa, Hidetoshi; Noble, Peter A.; El Fantroussi, Said; Kelly, John J.; Stahl, David A.

2002-01-01

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.

Genomic Analysis of Vaccine-Derived Poliovirus Strains in Stool Specimens by Combination of Full-Length PCR and Oligonucleotide Microarray Hybridization

PubMed Central

Laassri, Majid; Dragunsky, Eugenia; Enterline, Joan; Eremeeva, Tatiana; Ivanova, Olga; Lottenbach, Kathleen; Belshe, Robert; Chumakov, Konstantin

2005-01-01

Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5′ untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies. PMID:15956413

Under the influence of the active deodorant ingredient 4-hydroxy-3-methoxybenzyl alcohol, the skin bacterium Corynebacterium jeikeium moderately responds with differential gene expression.

PubMed

Brune, Iris; Becker, Anke; Paarmann, Daniel; Albersmeier, Andreas; Kalinowski, Jörn; Pühler, Alfred; Tauch, Andreas

2006-12-15

A 70mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Corynebacterium jeikeium, a skin bacterium that is predominantly present in the human axilla and involved in axillary odor formation. Oligonucleotides representing 100% of the predicted coding regions of the C. jeikeium K411 genome were designed and spotted in quadruplicate onto epoxy-coated glass slides. The quality of the printed microarray was demonstrated by co-hybridization with fluorescently labeled cDNA probes obtained from exponentially growing C. jeikeium cultures. Accordingly, genes detected with different intensities resulting in log(2) transformed ratios greater than 0.8 or smaller than -0.8 can be regarded as differentially expressed with a confidence level greater than 99%. In an application example, we measured global changes of gene expression during growth of C. jeikeium in the presence of different concentrations of the deodorant component 4-hydroxy-3-methoxybenzyl alcohol that is active in preventing body odor formation. Global expression profiling revealed that low concentrations of 4-hydroxy-3-methoxybenzyl alcohol (0.5 and 2.5mg/ml) had almost no detectable effect on the transcriptome of C. jeikeium. A slightly higher concentration of 4-hydroxy-3-methoxybenzyl alcohol (5mg/ml) resulted in differential expression of 95 genes, 86 of which showed an enhanced expression when compared to a control culture. Besides many genes encoding proteins that apparently participate in transcription and translation, the drug resistance determinant cmx and the predicted virulence factors sapA and sapD showed significantly enhanced expression levels. Differential expression of relevant genes was validated by real-time reverse transcription PCR assays.

Oligonucleotide microarray analysis of apoptosis induced by 15-methoxypinusolidic acid in microglial BV2 cells

PubMed Central

Choi, Y; Lim, SY; Jeong, HS; Koo, KA; Sung, SH; Kim, YC

2009-01-01

Background and purpose: We conducted a genome wide gene expression analysis to explore the biological aspects of 15-methoxypinusolidic acid (15-MPA) isolated from Biota orientalis and tried to confirm the suitability of 15-MPA as a therapeutic candidate for CNS injuries focusing on microglia. Experimental approach: Murine microglial BV2 cells were treated with 15-MPA, and their transcriptome was analysed by using oligonucleotide microarrays. Genes differentially expressed upon 15-MPA treatment were selected for RT-PCR (reverse transcription-polymerase chain reaction) analysis to confirm the gene expression. Inhibition of cell proliferation and induction of apoptosis by 15-MPA were examined by bromodeoxyuridine assay, Western blot analysis of poly-ADP-ribose polymerase and flow cytometry. Key results: A total of 514 genes were differentially expressed by 15-MPA treatment. Biological pathway analysis revealed that 15-MPA induced significant changes in expression of genes in the cell cycle pathway. Genes involved in growth arrest and DNA damage [gadd45α, gadd45γ and ddit3 (DNA damage-inducible transcript 3)] and cyclin-dependent kinase inhibitor (cdkn2b) were up-regulated, whereas genes involved in cell cycle progression (ccnd1, ccnd3 and ccne1), DNA replication (mcm4, orc1l and cdc6) and cell proliferation (fos and jun) were down-regulated. RT-PCR analysis for representative genes confirmed the expression levels. 15-MPA significantly reduced bromodeoxyuridine incorporation, increased poly-ADP-ribose polymerase cleavage and the number of apoptotic cells, indicating that 15-MPA induces apoptosis in BV2 cells. Conclusion and implications: 15-MPA induced apoptosis in murine microglial cells, presumably via inhibition of the cell cycle progression. As microglial activation is detrimental in CNS injuries, these data suggest a strong therapeutic potential of 15-MPA. PMID:19466985

Identifying Fishes through DNA Barcodes and Microarrays.

PubMed

Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N; Weber, Hannes; Blohm, Dietmar

2010-09-07

International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

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

PubMed

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

2009-10-27

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. 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. Gene ARMADA provides a highly adaptable, integrative, yet flexible tool which can be used for automated quality control, analysis, annotation and visualization of microarray data, constituting a starting point for further data interpretation and integration with numerous other tools.

Open-target sparse sensing of biological agents using DNA microarray

PubMed Central

2011-01-01

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

Using gene chips to identify organ-specific, smooth muscle responses to experimental diabetes: potential applications to urological diseases.

PubMed

Hipp, Jason D; Davies, Kelvin P; Tar, Moses; Valcic, Mira; Knoll, Abraham; Melman, Arnold; Christ, George J

2007-02-01

To identify early diabetes-related alterations in gene expression in bladder and erectile tissue that would provide novel diagnostic and therapeutic treatment targets to prevent, delay or ameliorate the ensuing bladder and erectile dysfunction. The RG-U34A rat GeneChip (Affymetrix Inc., Sunnyvale, CA, USA) oligonucleotide microarray (containing approximately 8799 genes) was used to evaluate gene expression in corporal and male bladder tissue excised from rats 1 week after confirmation of a diabetic state, but before demonstrable changes in organ function in vivo. A conservative analytical approach was used to detect alterations in gene expression, and gene ontology (GO) classifications were used to identify biological themes/pathways involved in the aetiology of the organ dysfunction. In all, 320 and 313 genes were differentially expressed in bladder and corporal tissue, respectively. GO analysis in bladder tissue showed prominent increases in biological pathways involved in cell proliferation, metabolism, actin cytoskeleton and myosin, as well as decreases in cell motility, and regulation of muscle contraction. GO analysis in corpora showed increases in pathways related to ion channel transport and ion channel activity, while there were decreases in collagen I and actin genes. The changes in gene expression in these initial experiments are consistent with the pathophysiological characteristics of the bladder and erectile dysfunction seen later in the diabetic disease process. Thus, the observed changes in gene expression might be harbingers or biomarkers of impending organ dysfunction, and could provide useful diagnostic and therapeutic targets for a variety of progressive urological diseases/conditions (i.e. lower urinary tract symptoms related to benign prostatic hyperplasia, erectile dysfunction, etc.).

European Thyroid Association Guidelines regarding Thyroid Nodule Molecular Fine-Needle Aspiration Cytology Diagnostics.

PubMed

Paschke, Ralf; Cantara, Silvia; Crescenzi, Anna; Jarzab, Barbara; Musholt, Thomas J; Sobrinho Simoes, Manuel

2017-07-01

Molecular fine-needle aspiration (FNA) cytology diagnostics has the potential to address the inherent limitation of FNA cytology which is an indeterminate (atypia of undetermined significance/follicular lesion of undetermined significance follicular neoplasm) cytology. Because of the emerging role of molecular FNA cytology diagnostics, the European Thyroid Association convened a panel of international experts to review methodological aspects, indications, results, and limitations of molecular FNA cytology diagnostics. The panel reviewed the evidence for the diagnostic value of mutation panel assessment (including at least BRAF , NRAS , HRAS , KRAS , PAX8/PPARG , RET/PTC ) of targeted next generation sequencing and of a microarray gene expression classifier (GEC) test in the diagnostic assessment of an indeterminate cytology thyroid nodule. Moreover, possible surgical consequences of molecular FNA diagnostic results of thyroid nodules and the evidence that analysis of a molecular FNA diagnostic panel of somatic mutations or a microarray GEC test can alter the follow-up were reviewed. Molecular tests may help clinicians to drive patient care and the surgical decision if the analysis is performed in specialized laboratories. These molecular tests require standardization of performance characteristics and appropriate calibration as well as analytic validation before clinical interpretation.

European Thyroid Association Guidelines regarding Thyroid Nodule Molecular Fine-Needle Aspiration Cytology Diagnostics

PubMed Central

Paschke, Ralf; Cantara, Silvia; Crescenzi, Anna; Jarzab, Barbara; Musholt, Thomas J.; Sobrinho Simoes, Manuel

2017-01-01

Molecular fine-needle aspiration (FNA) cytology diagnostics has the potential to address the inherent limitation of FNA cytology which is an indeterminate (atypia of undetermined significance/follicular lesion of undetermined significance follicular neoplasm) cytology. Because of the emerging role of molecular FNA cytology diagnostics, the European Thyroid Association convened a panel of international experts to review methodological aspects, indications, results, and limitations of molecular FNA cytology diagnostics. The panel reviewed the evidence for the diagnostic value of mutation panel assessment (including at least BRAF, NRAS, HRAS, KRAS, PAX8/PPARG, RET/PTC) of targeted next generation sequencing and of a microarray gene expression classifier (GEC) test in the diagnostic assessment of an indeterminate cytology thyroid nodule. Moreover, possible surgical consequences of molecular FNA diagnostic results of thyroid nodules and the evidence that analysis of a molecular FNA diagnostic panel of somatic mutations or a microarray GEC test can alter the follow-up were reviewed. Molecular tests may help clinicians to drive patient care and the surgical decision if the analysis is performed in specialized laboratories. These molecular tests require standardization of performance characteristics and appropriate calibration as well as analytic validation before clinical interpretation. PMID:28785538

Living Cell Microarrays: An Overview of Concepts

PubMed Central

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

2016-01-01

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

cDNA Microarray Screening in Food Safety

PubMed Central

ROY, SASHWATI; SEN, CHANDAN K

2009-01-01

The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests. PMID:16466843

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…

probeBase—an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016

PubMed Central

Greuter, Daniel; Loy, Alexander; Horn, Matthias; Rattei, Thomas

2016-01-01

probeBase http://www.probebase.net is a manually maintained and curated database of rRNA-targeted oligonucleotide probes and primers. Contextual information and multiple options for evaluating in silico hybridization performance against the most recent rRNA sequence databases are provided for each oligonucleotide entry, which makes probeBase an important and frequently used resource for microbiology research and diagnostics. Here we present a major update of probeBase, which was last featured in the NAR Database Issue 2007. This update describes a complete remodeling of the database architecture and environment to accommodate computationally efficient access. Improved search functions, sequence match tools and data output now extend the opportunities for finding suitable hierarchical probe sets that target an organism or taxon at different taxonomic levels. To facilitate the identification of complementary probe sets for organisms represented by short rRNA sequence reads generated by amplicon sequencing or metagenomic analysis with next generation sequencing technologies such as Illumina and IonTorrent, we introduce a novel tool that recovers surrogate near full-length rRNA sequences for short query sequences and finds matching oligonucleotides in probeBase. PMID:26586809

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

PubMed Central

2010-01-01

Background The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. Findings We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. Conclusions A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis. PMID:20565839

Detecting novel genes with sparse arrays

PubMed Central

Haiminen, Niina; Smit, Bart; Rautio, Jari; Vitikainen, Marika; Wiebe, Marilyn; Martinez, Diego; Chee, Christine; Kunkel, Joe; Sanchez, Charles; Nelson, Mary Anne; Pakula, Tiina; Saloheimo, Markku; Penttilä, Merja; Kivioja, Teemu

2014-01-01

Species-specific genes play an important role in defining the phenotype of an organism. However, current gene prediction methods can only efficiently find genes that share features such as sequence similarity or general sequence characteristics with previously known genes. Novel sequencing methods and tiling arrays can be used to find genes without prior information and they have demonstrated that novel genes can still be found from extensively studied model organisms. Unfortunately, these methods are expensive and thus are not easily applicable, e.g., to finding genes that are expressed only in very specific conditions. We demonstrate a method for finding novel genes with sparse arrays, applying it on the 33.9 Mb genome of the filamentous fungus Trichoderma reesei. Our computational method does not require normalisations between arrays and it takes into account the multiple-testing problem typical for analysis of microarray data. In contrast to tiling arrays, that use overlapping probes, only one 25mer microarray oligonucleotide probe was used for every 100 b. Thus, only relatively little space on a microarray slide was required to cover the intergenic regions of a genome. The analysis was done as a by-product of a conventional microarray experiment with no additional costs. We found at least 23 good candidates for novel transcripts that could code for proteins and all of which were expressed at high levels. Candidate genes were found to neighbour ire1 and cre1 and many other regulatory genes. Our simple, low-cost method can easily be applied to finding novel species-specific genes without prior knowledge of their sequence properties. PMID:20691772

Hybrid genetic algorithm-neural network: feature extraction for unpreprocessed microarray data.

PubMed

Tong, Dong Ling; Schierz, Amanda C

2011-09-01

Suitable techniques for microarray analysis have been widely researched, particularly for the study of marker genes expressed to a specific type of cancer. Most of the machine learning methods that have been applied to significant gene selection focus on the classification ability rather than the selection ability of the method. These methods also require the microarray data to be preprocessed before analysis takes place. The objective of this study is to develop a hybrid genetic algorithm-neural network (GANN) model that emphasises feature selection and can operate on unpreprocessed microarray data. The GANN is a hybrid model where the fitness value of the genetic algorithm (GA) is based upon the number of samples correctly labelled by a standard feedforward artificial neural network (ANN). The model is evaluated by using two benchmark microarray datasets with different array platforms and differing number of classes (a 2-class oligonucleotide microarray data for acute leukaemia and a 4-class complementary DNA (cDNA) microarray dataset for SRBCTs (small round blue cell tumours)). The underlying concept of the GANN algorithm is to select highly informative genes by co-evolving both the GA fitness function and the ANN weights at the same time. The novel GANN selected approximately 50% of the same genes as the original studies. This may indicate that these common genes are more biologically significant than other genes in the datasets. The remaining 50% of the significant genes identified were used to build predictive models and for both datasets, the models based on the set of genes extracted by the GANN method produced more accurate results. The results also suggest that the GANN method not only can detect genes that are exclusively associated with a single cancer type but can also explore the genes that are differentially expressed in multiple cancer types. The results show that the GANN model has successfully extracted statistically significant genes from the unpreprocessed microarray data as well as extracting known biologically significant genes. We also show that assessing the biological significance of genes based on classification accuracy may be misleading and though the GANN's set of extra genes prove to be more statistically significant than those selected by other methods, a biological assessment of these genes is highly recommended to confirm their functionality. Copyright © 2011 Elsevier B.V. All rights reserved.

Broad spectrum microarray for fingerprint-based bacterial species identification

PubMed Central

2010-01-01

Background Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. Results A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. Conclusions These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups. PMID:20163710

Multiclassifier information fusion methods for microarray pattern recognition

NASA Astrophysics Data System (ADS)

Braun, Jerome J.; Glina, Yan; Judson, Nicholas; Herzig-Marx, Rachel

2004-04-01

This paper addresses automatic recognition of microarray patterns, a capability that could have a major significance for medical diagnostics, enabling development of diagnostic tools for automatic discrimination of specific diseases. The paper presents multiclassifier information fusion methods for microarray pattern recognition. The input space partitioning approach based on fitness measures that constitute an a-priori gauging of classification efficacy for each subspace is investigated. Methods for generation of fitness measures, generation of input subspaces and their use in the multiclassifier fusion architecture are presented. In particular, two-level quantification of fitness that accounts for the quality of each subspace as well as the quality of individual neighborhoods within the subspace is described. Individual-subspace classifiers are Support Vector Machine based. The decision fusion stage fuses the information from mulitple SVMs along with the multi-level fitness information. Final decision fusion stage techniques, including weighted fusion as well as Dempster-Shafer theory based fusion are investigated. It should be noted that while the above methods are discussed in the context of microarray pattern recognition, they are applicable to a broader range of discrimination problems, in particular to problems involving a large number of information sources irreducible to a low-dimensional feature space.

Chromosomal Microarray With Clinical Diagnostic Utility in Children With Developmental Delay or Intellectual Disability.

PubMed

Lee, Jin Sook; Hwang, Hee; Kim, Soo Yeon; Kim, Ki Joong; Choi, Jin Sun; Woo, Mi Jung; Choi, Young Min; Jun, Jong Kwan; Lim, Byung Chan; Chae, Jong Hee

2018-09-01

Chromosomal microarray (CMA) testing is a first-tier test for patients with developmental delay, autism, or congenital anomalies. It increases diagnostic yield for patients with developmental delay or intellectual disability. In some countries, including Korea, CMA testing is not yet implemented in clinical practice. We assessed the diagnostic utility of CMA testing in a large cohort of patients with developmental delay or intellectual disability in Korea. We conducted a genome-wide microarray analysis of 649 consecutive patients with developmental delay or intellectual disability at the Seoul National University Children's Hospital. Medical records were reviewed retrospectively. Pathogenicity of detected copy number variations (CNVs) was evaluated by referencing previous reports or parental testing using FISH or quantitative PCR. We found 110 patients to have pathogenic CNVs, which included 100 deletions and 31 duplications of 270 kb to 30 Mb. The diagnostic yield was 16.9%, demonstrating the diagnostic utility of CMA testing in clinic. Parental testing was performed in 66 patients, 86.4% of which carried de novo CNVs. In eight patients, pathogenic CNVs were inherited from healthy parents with a balanced translocation, and genetic counseling was provided to these families. We verified five rarely reported deletions on 2p21p16.3, 3p21.31, 10p11.22, 14q24.2, and 21q22.13. This study demonstrated the clinical utility of CMA testing in the genetic diagnosis of patients with developmental delay or intellectual disability. CMA testing should be included as a clinical diagnostic test for all children with developmental delay or intellectual disability. © The Korean Society for Laboratory Medicine.

Experience With Rapid Microarray-Based Diagnostic Technology and Antimicrobial Stewardship for Patients With Gram-Positive Bacteremia.

PubMed

Neuner, Elizabeth A; Pallotta, Andrea M; Lam, Simon W; Stowe, David; Gordon, Steven M; Procop, Gary W; Richter, Sandra S

2016-11-01

OBJECTIVE To describe the impact of rapid diagnostic microarray technology and antimicrobial stewardship for patients with Gram-positive blood cultures. DESIGN Retrospective pre-intervention/post-intervention study. SETTING A 1,200-bed academic medical center. PATIENTS Inpatients with blood cultures positive for Staphylococcus aureus, Enterococcus faecalis, E. faecium, Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. anginosus, Streptococcus spp., and Listeria monocytogenes during the 6 months before and after implementation of Verigene Gram-positive blood culture microarray (BC-GP) with an antimicrobial stewardship intervention. METHODS Before the intervention, no rapid diagnostic technology was used or antimicrobial stewardship intervention was undertaken, except for the use of peptide nucleic acid fluorescent in situ hybridization and MRSA agar to identify staphylococcal isolates. After the intervention, all Gram-positive blood cultures underwent BC-GP microarray and the antimicrobial stewardship intervention consisting of real-time notification and pharmacist review. RESULTS In total, 513 patients with bacteremia were included in this study: 280 patients with S. aureus, 150 patients with enterococci, 82 patients with stretococci, and 1 patient with L. monocytogenes. The number of antimicrobial switches was similar in the pre-BC-GP (52%; 155 of 300) and post-BC-GP (50%; 107 of 213) periods. The time to antimicrobial switch was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 48±41 hours versus 75±46 hours, respectively (P<.001). The most common antimicrobial switch was de-escalation and time to de-escalation, was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 53±41 hours versus 82±48 hours, respectively (P<.001). There was no difference in mortality or hospital length of stay as a result of the intervention. CONCLUSIONS The combination of a rapid microarray diagnostic test with an antimicrobial stewardship intervention improved time to antimicrobial switch, especially time to de-escalation to optimal therapy, in patients with Gram-positive blood cultures. Infect Control Hosp Epidemiol 2016;1-6.

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

PubMed

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

2015-10-01

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

Gene expression analysis of hypersensitivity to mosquito bite, chronic active EBV infection and NK/T-lymphoma/leukemia.

PubMed

Washio, Kana; Oka, Takashi; Abdalkader, Lamia; Muraoka, Michiko; Shimada, Akira; Oda, Megumi; Sato, Hiaki; Takata, Katsuyoshi; Kagami, Yoshitoyo; Shimizu, Norio; Kato, Seiichi; Kimura, Hiroshi; Nishizaki, Kazunori; Yoshino, Tadashi; Tsukahara, Hirokazu

2017-11-01

The human herpes virus, Epstein-Barr virus (EBV), is a known oncogenic virus and plays important roles in life-threatening T/NK-cell lymphoproliferative disorders (T/NK-cell LPD) such as hypersensitivity to mosquito bite (HMB), chronic active EBV infection (CAEBV), and NK/T-cell lymphoma/leukemia. During the clinical courses of HMB and CAEBV, patients frequently develop malignant lymphomas and the diseases passively progress sequentially. In the present study, gene expression of CD16 (-) CD56 (+) -, EBV (+) HMB, CAEBV, NK-lymphoma, and NK-leukemia cell lines, which were established from patients, was analyzed using oligonucleotide microarrays and compared to that of CD56 bright CD16 dim/- NK cells from healthy donors. Principal components analysis showed that CAEBV and NK-lymphoma cells were relatively closely located, indicating that they had similar expression profiles. Unsupervised hierarchal clustering analyses of microarray data and gene ontology analysis revealed specific gene clusters and identified several candidate genes responsible for disease that can be used to discriminate each category of NK-LPD and NK-cell lymphoma/leukemia.

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

PubMed Central

Kamalakaran, Sitharthan; Kendall, Jude; Zhao, Xiaoyue; Tang, Chunlao; Khan, Sohail; Ravi, Kandasamy; Auletta, Theresa; Riggs, Michael; Wang, Yun; Helland, Åslaug; Naume, Bjørn; Dimitrova, Nevenka; Børresen-Dale, Anne-Lise; Hicks, Jim; Lucito, Robert

2009-01-01

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species. PMID:19474344

Oligonucleotide Array for Identification and Detection of Pythium Species†

PubMed Central

Tambong, J. T.; de Cock, A. W. A. M.; Tinker, N. A.; Lévesque, C. A.

2006-01-01

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies. PMID:16597974

A novel 4p16.3 microduplication distal to WHSC1 and WHSC2 characterized by oligonucleotide array with new phenotypic features.

PubMed

Cyr, Andrew B; Nimmakayalu, Manjunath; Longmuir, Susannah Q; Patil, Shivanand R; Keppler-Noreuil, Kim M; Shchelochkov, Oleg A

2011-09-01

Larger imbalances on chromosome 4p in the form of deletions associated with Wolf-Hirschhorn syndrome (WHS) and duplications of chromosome 4p have a defined clinical phenotype. The critical region for both these clinical disorders has been narrowed based on the genotype-phenotype correlations. However, cryptic rearrangements in this region have been reported infrequently. We report on a male patient with a microduplication of chromosome 4p, who presents with findings of macrocephaly, irregular iris pigmentation-heterochromia, and preserved linear growth in addition to overlapping features of trisomy 4p such as seizures, delayed psychomotor development, and dysmorphic features including prominent glabella, low-set ears, and short neck. Using a high-density oligonucleotide microarray, we have identified a novel submicroscopic duplication involving dosage sensitive genes TACC3, FGFR3, and LETM1. The microduplication did not involve WHSC1 and WHSC2 which are considered in the critical region for WHS and trisomy 4p. This patient's presentation and genomic findings help further delineate clinical significance of re-arrangements in the 4p16 region without the involvement of WHS critical region. Copyright © 2011 Wiley-Liss, Inc.

Identification of differentially regulated transcripts in mouse striatum following methamphetamine treatment--an oligonucleotide microarray approach.

PubMed

Thomas, David M; Francescutti-Verbeem, Dina M; Liu, Xiuli; Kuhn, Donald M

2004-01-01

Methamphetamine is an addictive drug of abuse that can produce neurotoxic effects in dopamine nerve endings of the striatum. The purpose of this study was to identify new genes that may play a role in the highly complex cascade of events associated with methamphetamine intoxication. Using Affymetrix oligonucleotide arrays, 12 488 genes were simultaneously interrogated and there were 152 whose expression levels were changed following methamphetamine treatment. The genes are grouped into broad functional categories with inflammatory/immune response elements, receptor/signal transduction components and ion channel/transport proteins among the most populated. Many genes within these categories can be linked to ion regulation and apoptosis, both of which have been implicated in methamphetamine toxicity, and numerous factors associated with microglial activation emerged with significant changes in expression. For example, brain-derived neurotrophic factor (BDNF), chemokine (C-C) receptor 6 (CCr6) and numerous chemokine transcripts were increased or decreased in expression more than 2.8-fold. These results point to activated microglia as a potential source of the reactive oxygen/nitrogen species and cytokines that have been previously associated with methamphetamine toxicity and other neurotoxic conditions.

Applications of nanotechnology, next generation sequencing and microarrays in biomedical research.

PubMed

Elingaramil, Sauli; Li, Xiaolong; He, Nongyue

2013-07-01

Next-generation sequencing technologies, microarrays and advances in bio nanotechnology have had an enormous impact on research within a short time frame. This impact appears certain to increase further as many biomedical institutions are now acquiring these prevailing new technologies. Beyond conventional sampling of genome content, wide-ranging applications are rapidly evolving for next-generation sequencing, microarrays and nanotechnology. To date, these technologies have been applied in a variety of contexts, including whole-genome sequencing, targeted re sequencing and discovery of transcription factor binding sites, noncoding RNA expression profiling and molecular diagnostics. This paper thus discusses current applications of nanotechnology, next-generation sequencing technologies and microarrays in biomedical research and highlights the transforming potential these technologies offer.

Advances in cell-free protein array methods.

PubMed

Yu, Xiaobo; Petritis, Brianne; Duan, Hu; Xu, Danke; LaBaer, Joshua

2018-01-01

Cell-free protein microarrays represent a special form of protein microarray which display proteins made fresh at the time of the experiment, avoiding storage and denaturation. They have been used increasingly in basic and translational research over the past decade to study protein-protein interactions, the pathogen-host relationship, post-translational modifications, and antibody biomarkers of different human diseases. Their role in the first blood-based diagnostic test for early stage breast cancer highlights their value in managing human health. Cell-free protein microarrays will continue to evolve to become widespread tools for research and clinical management. Areas covered: We review the advantages and disadvantages of different cell-free protein arrays, with an emphasis on the methods that have been studied in the last five years. We also discuss the applications of each microarray method. Expert commentary: Given the growing roles and impact of cell-free protein microarrays in research and medicine, we discuss: 1) the current technical and practical limitations of cell-free protein microarrays; 2) the biomarker discovery and verification pipeline using protein microarrays; and 3) how cell-free protein microarrays will advance over the next five years, both in their technology and applications.

Is this the real time for genomics?

PubMed

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

2014-01-01

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

Parallel gene analysis with allele-specific padlock probes and tag microarrays

PubMed Central

Banér, Johan; Isaksson, Anders; Waldenström, Erik; Jarvius, Jonas; Landegren, Ulf; Nilsson, Mats

2003-01-01

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes. PMID:12930977

Genome-wide polymorphisms and development of a microarray platform to detect genetic variations in Plasmodium yoelii.

PubMed

Nair, Sethu C; Pattaradilokrat, Sittiporn; Zilversmit, Martine M; Dommer, Jennifer; Nagarajan, Vijayaraj; Stephens, Melissa T; Xiao, Wenming; Tan, John C; Su, Xin-Zhuan

2014-01-01

The rodent malaria parasite Plasmodium yoelii is an important model for studying malaria immunity and pathogenesis. One approach for studying malaria disease phenotypes is genetic mapping, which requires typing a large number of genetic markers from multiple parasite strains and/or progeny from genetic crosses. Hundreds of microsatellite (MS) markers have been developed to genotype the P. yoelii genome; however, typing a large number of MS markers can be labor intensive, time consuming, and expensive. Thus, development of high-throughput genotyping tools such as DNA microarrays that enable rapid and accurate large-scale genotyping of the malaria parasite will be highly desirable. In this study, we sequenced the genomes of two P. yoelii strains (33X and N67) and obtained a large number of single nucleotide polymorphisms (SNPs). Based on the SNPs obtained, we designed sets of oligonucleotide probes to develop a microarray that could interrogate ∼11,000 SNPs across the 14 chromosomes of the parasite in a single hybridization. Results from hybridizations of DNA samples of five P. yoelii strains or cloned lines (17XNL, YM, 33X, N67 and N67C) and two progeny from a genetic cross (N67×17XNL) to the microarray showed that the array had a high call rate (∼97%) and accuracy (99.9%) in calling SNPs, providing a simple and reliable tool for typing the P. yoelii genome. Our data show that the P. yoelii genome is highly polymorphic, although isogenic pairs of parasites were also detected. Additionally, our results indicate that the 33X parasite is a progeny of 17XNL (or YM) and an unknown parasite. The highly accurate and reliable microarray developed in this study will greatly facilitate our ability to study the genetic basis of important traits and the disease it causes. Published by Elsevier B.V.

Improvement in the amine glass platform by bubbling method for a DNA microarray

PubMed Central

Jee, Seung Hyun; Kim, Jong Won; Lee, Ji Hyeong; Yoon, Young Soo

2015-01-01

A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool. PMID:26468293

Improvement in the amine glass platform by bubbling method for a DNA microarray.

PubMed

Jee, Seung Hyun; Kim, Jong Won; Lee, Ji Hyeong; Yoon, Young Soo

2015-01-01

A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool.

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

PubMed

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

2016-12-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2012-01-01

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

PhyloFlu, a DNA microarray for determining the phylogenetic origin of influenza A virus gene segments and the genomic fingerprint of viral strains.

PubMed

Paulin, Luis F; de los D Soto-Del Río, María; Sánchez, Iván; Hernández, Jesús; Gutiérrez-Ríos, Rosa M; López-Martínez, Irma; Wong-Chew, Rosa M; Parissi-Crivelli, Aurora; Isa, P; López, Susana; Arias, Carlos F

2014-03-01

Recent evidence suggests that most influenza A virus gene segments can contribute to the pathogenicity of the virus. In this regard, the hemagglutinin (HA) subtype of the circulating strains has been closely surveyed, but the reassortment of internal gene segments is usually not monitored as a potential source of an increased pathogenicity. In this work, an oligonucleotide DNA microarray (PhyloFlu) designed to determine the phylogenetic origins of the eight segments of the influenza virus genome was constructed and validated. Clades were defined for each segment and also for the 16 HA and 9 neuraminidase (NA) subtypes. Viral genetic material was amplified by reverse transcription-PCR (RT-PCR) with primers specific to the conserved 5' and 3' ends of the influenza A virus genes, followed by PCR amplification with random primers and Cy3 labeling. The microarray unambiguously determined the clades for all eight influenza virus genes in 74% (28/38) of the samples. The microarray was validated with reference strains from different animal origins, as well as from human, swine, and avian viruses from field or clinical samples. In most cases, the phylogenetic clade of each segment defined its animal host of origin. The genomic fingerprint deduced by the combined information of the individual clades allowed for the determination of the time and place that strains with the same genomic pattern were previously reported. PhyloFlu is useful for characterizing and surveying the genetic diversity and variation of animal viruses circulating in different environmental niches and for obtaining a more detailed surveillance and follow up of reassortant events that can potentially modify virus pathogenicity.

Near-isogenic cotton germplasm lines that differ in fiber-bundle strength have temporal differences in fiber gene expression patterns as revealed by comparative high-throughput profiling.

PubMed

Hinchliffe, Doug J; Meredith, William R; Yeater, Kathleen M; Kim, Hee Jin; Woodward, Andrew W; Chen, Z Jeffrey; Triplett, Barbara A

2010-05-01

Gene expression profiles of developing cotton (Gossypium hirsutum L.) fibers from two near-isogenic lines (NILs) that differ in fiber-bundle strength, short-fiber content, and in fewer than two genetic loci were compared using an oligonucleotide microarray. Fiber gene expression was compared at five time points spanning fiber elongation and secondary cell wall (SCW) biosynthesis. Fiber samples were collected from field plots in a randomized, complete block design, with three spatially distinct biological replications for each NIL at each time point. Microarray hybridizations were performed in a loop experimental design that allowed comparisons of fiber gene expression profiles as a function of time between the two NILs. Overall, developmental expression patterns revealed by the microarray experiment agreed with previously reported cotton fiber gene expression patterns for specific genes. Additionally, genes expressed coordinately with the onset of SCW biosynthesis in cotton fiber correlated with gene expression patterns of other SCW-producing plant tissues. Functional classification and enrichment analysis of differentially expressed genes between the two NILs revealed that genes associated with SCW biosynthesis were significantly up-regulated in fibers of the high-fiber quality line at the transition stage of cotton fiber development. For independent corroboration of the microarray results, 15 genes were selected for quantitative reverse transcription PCR analysis of fiber gene expression. These analyses, conducted over multiple field years, confirmed the temporal difference in fiber gene expression between the two NILs. We hypothesize that the loci conferring temporal differences in fiber gene expression between the NILs are important regulatory sequences that offer the potential for more targeted manipulation of cotton fiber quality.

Calibration and assessment of channel-specific biases in microarray data with extended dynamical range.

PubMed

Bengtsson, Henrik; Jönsson, Göran; Vallon-Christersson, Johan

2004-11-12

Non-linearities in observed log-ratios of gene expressions, also known as intensity dependent log-ratios, can often be accounted for by global biases in the two channels being compared. Any step in a microarray process may introduce such offsets and in this article we study the biases introduced by the microarray scanner and the image analysis software. By scanning the same spotted oligonucleotide microarray at different photomultiplier tube (PMT) gains, we have identified a channel-specific bias present in two-channel microarray data. For the scanners analyzed it was in the range of 15-25 (out of 65,535). The observed bias was very stable between subsequent scans of the same array although the PMT gain was greatly adjusted. This indicates that the bias does not originate from a step preceding the scanner detector parts. The bias varies slightly between arrays. When comparing estimates based on data from the same array, but from different scanners, we have found that different scanners introduce different amounts of bias. So do various image analysis methods. We propose a scanning protocol and a constrained affine model that allows us to identify and estimate the bias in each channel. Backward transformation removes the bias and brings the channels to the same scale. The result is that systematic effects such as intensity dependent log-ratios are removed, but also that signal densities become much more similar. The average scan, which has a larger dynamical range and greater signal-to-noise ratio than individual scans, can then be obtained. The study shows that microarray scanners may introduce a significant bias in each channel. Such biases have to be calibrated for, otherwise systematic effects such as intensity dependent log-ratios will be observed. The proposed scanning protocol and calibration method is simple to use and is useful for evaluating scanner biases or for obtaining calibrated measurements with extended dynamical range and better precision. The cross-platform R package aroma, which implements all described methods, is available for free from http://www.maths.lth.se/bioinformatics/.

Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2'-deoxycytidine treatment and oligonucleotide microarray.

PubMed

Yamashita, Satoshi; Tsujino, Yoshimi; Moriguchi, Kazuki; Tatematsu, Masae; Ushijima, Toshikazu

2006-01-01

To identify novel methylation-silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome-wide search for genes upregulated by treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC). After 5-aza-dC treatment of a gastric cancer cell line (AGS) 579 genes were upregulated 16-fold or more, using an oligonucleotide microarray with 39,000 genes. From these genes, we selected 44 known genes on autosomes whose silencing in gastric cancer has not been reported. Thirty-two of these had CpG islands (CGI) in their putative promoter regions, and all of the CGI were methylated in AGS, giving an estimated number of 421+/-75 (95% confidence interval) methylation-silenced genes. Additionally, we analyzed the methylation status of 16 potential tumor-related genes with promoter CGI that were upregulated four-fold or more, and 14 of these were methylated in AGS. Methylation status of the 32 randomly selected and 16 potential tumor-related genes was analyzed in 10 primary gastric cancers, and 42 genes (ABHD9, ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, ULBP2, WIF1, ZNF177 and ZNF559) were methylated in at least one primary gastric cancer. A metastasis suppressor gene, MTSS1, was located in a genomic region with frequent loss of heterozygosity (8q22), and was expressed abundantly in the normal gastric mucosa, suggesting its role in gastric carcinogenesis. (Cancer Sci 2006; 97: 64 -71). (Cancer Sci 2006; 97: 64 -71).

High Boron-loaded DNA-Oligomers as Potential Boron Neutron Capture Therapy and Antisense Oligonucleotide Dual-Action Anticancer Agents.

PubMed

Kaniowski, Damian; Ebenryter-Olbińska, Katarzyna; Sobczak, Milena; Wojtczak, Błażej; Janczak, Sławomir; Leśnikowski, Zbigniew J; Nawrot, Barbara

2017-08-23

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted "click" conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400-2650 cm -1 . The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 μM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

A perspective on microarrays: current applications, pitfalls, and potential uses

PubMed Central

Jaluria, Pratik; Konstantopoulos, Konstantinos; Betenbaugh, Michael; Shiloach, Joseph

2007-01-01

With advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. In fact, fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefiting from its use. However, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. Although, microarray technology is relatively simple, conceptually, its practice does require careful planning and detailed understanding of the limitations inherently present. Without these considerations, it can be exceedingly difficult to ascertain valuable information from microarray data. Therefore, this text aims to outline key features in microarray technology, paying particular attention to current applications as outlined in recent publications, experimental design, statistical methods, and potential uses. Furthermore, this review is not meant to be comprehensive, but rather substantive; highlighting important concepts and detailing steps necessary to conduct and interpret microarray experiments. Collectively, the information included in this text will highlight the versatility of microarray technology and provide a glimpse of what the future may hold. PMID:17254338

Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage

PubMed Central

Dehne, T.; Lindahl, A.; Brittberg, M.; Pruss, A.; Ringe, J.; Sittinger, M.; Karlsson, C.

2012-01-01

Objective: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. Methods: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. Results: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca2+/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. Conclusions: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca2+/WNT pathway was activated in OA cartilage. PMID:26069618

A microarray for assessing transcription from pelagic marine microbial taxa

PubMed Central

Shilova, Irina N; Robidart, Julie C; James Tripp, H; Turk-Kubo, Kendra; Wawrik, Boris; Post, Anton F; Thompson, Anne W; Ward, Bess; Hollibaugh, James T; Millard, Andy; Ostrowski, Martin; J Scanlan, David; Paerl, Ryan W; Stuart, Rhona; Zehr, Jonathan P

2014-01-01

Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world's oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions. PMID:24477198

A microarray analysis of potential genes underlying the neurosensitivity of mice to propofol.

PubMed

Lowes, Damon A; Galley, Helen F; Lowe, Peter R; Rikke, Brad A; Johnson, Thomas E; Webster, Nigel R

2005-09-01

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and "BTB (POZ) domain containing 1," whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

Detection of microbial communities in continuous and discontinuous membrane bioreactor using high-density oligonucleotide Microarray

NASA Astrophysics Data System (ADS)

Duan, Liang; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W.

2010-11-01

This study compared the whole composition of microbial communities in continuous-flow (MBR) and batch-fed (discontinuous) (MSBR) aerobic membrane bioreactors using high-density universal 16S rRNA Microarray. The array includes 506,944 probes targeted to 8935 clusters in 16S rRNA gene sequences. The Microarray results showed that both MBR and MSBR had high microbial diversity. 1126 and 1002 bacterial subfamilies were detected and can separate as 37 and 32 phyla in MBR and MSBR, respectively. Proteobacteria was the predominant phylum, 703 and 597 subfamilies were found in two systems, which constituted 62.4% and 59.6% of the whole bacteria. Gamma- and Alpha-were the dominant classes in Proteobacteria. It occupied 38.1% and 26.3%, 31.2% and 39.2% for MBR and MSBR, respectively. Bacteroidetes, Firmicutes and Actinobacteria were the subdominant groups, occupying around 9.4% and 7.6%, 6.1% and 6.5%, 6.0% and 9.0% of the total bacteria in two reactors. Some bacterial groups such as Acidobacteria, Chloroflexi, Cyanobacteria, Verrucomicrobia and Spirochaetes also found more than 15 subfamilies. All the results indicated that the MBR system had more bacteria community diversity than MSBR's. Moreover, it was very interested that MBR and MSBR had almost the same bacterial composition except Enterobacteriaceae. 63 OTUs of Enterobacteriaceae were detected in MBR, while just 10 OTUs were found in MSBR. That's one of the reasons leading to the difference of the bacterial diversity between two bioreactors.

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

PubMed Central

List, Claudia; Qi, Weihong; Maag, Eva; Gottstein, Bruno; Müller, Norbert; Felger, Ingrid

2010-01-01

Background Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. Methodology/Principal Findings Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. Conclusions/Significance This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens. PMID:20689813

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

PubMed

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

2016-12-19

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

Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

NASA Astrophysics Data System (ADS)

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

2011-06-01

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

Dynamic, electronically switchable surfaces for membrane protein microarrays.

PubMed

Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

2006-02-01

Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

Automation of complex assays: pharmacogenetics of warfarin dosing.

PubMed

Wu, Whei-Kuo; Hujsak, Paul G; Kureshy, Fareed

2007-10-01

AutoGenomics, Inc. (Carlsbad, CA, USA) have developed a multiplex microarray assay for genotyping both VKORC1 and CYP2C9 using the INFINITI(™) Analyzer. Multiple alleles in each DNA sample are analyzed by polymerase chain reaction amplification, followed by detection primer extension using the INFINITI Analyzer. The INFINITI Analyzer performs single-nucleotide polymorphism (SNP) analysis using universal oligonucleotides immobilized on the biochip. To genotype broader ethnic groups, genomic DNA from whole blood was tested for nine SNPs for VKORC1 and six for CYP2C9 genotypes. Information related to all 15 SNPs is needed to determine dosing of population of diverse ethnic origin. The INFINITI system provides genotyping information for same day dosing of warfarin.

Animal Viruses Probe dataset (AVPDS) for microarray-based diagnosis and identification of viruses.

PubMed

Yadav, Brijesh S; Pokhriyal, Mayank; Vasishtha, Dinesh P; Sharma, Bhaskar

2014-03-01

AVPDS (Animal Viruses Probe dataset) is a dataset of virus-specific and conserve oligonucleotides for identification and diagnosis of viruses infecting animals. The current dataset contain 20,619 virus specific probes for 833 viruses and their subtypes and 3,988 conserved probes for 146 viral genera. Dataset of virus specific probe has been divided into two fields namely virus name and probe sequence. Similarly conserved probes for virus genera table have genus, and subgroup within genus name and probe sequence. The subgroup within genus is artificially divided subgroups with no taxonomic significance and contains probes which identifies viruses in that specific subgroup of the genus. Using this dataset we have successfully diagnosed the first case of Newcastle disease virus in sheep and reported a mixed infection of Bovine viral diarrhea and Bovine herpesvirus in cattle. These dataset also contains probes which cross reacts across species experimentally though computationally they meet specifications. These probes have been marked. We hope that this dataset will be useful in microarray-based detection of viruses. The dataset can be accessed through the link https://dl.dropboxusercontent.com/u/94060831/avpds/HOME.html.

SaDA: From Sampling to Data Analysis—An Extensible Open Source Infrastructure for Rapid, Robust and Automated Management and Analysis of Modern Ecological High-Throughput Microarray Data

PubMed Central

Singh, Kumar Saurabh; Thual, Dominique; Spurio, Roberto; Cannata, Nicola

2015-01-01

One of the most crucial characteristics of day-to-day laboratory information management is the collection, storage and retrieval of information about research subjects and environmental or biomedical samples. An efficient link between sample data and experimental results is absolutely important for the successful outcome of a collaborative project. Currently available software solutions are largely limited to large scale, expensive commercial Laboratory Information Management Systems (LIMS). Acquiring such LIMS indeed can bring laboratory information management to a higher level, but most of the times this requires a sufficient investment of money, time and technical efforts. There is a clear need for a light weighted open source system which can easily be managed on local servers and handled by individual researchers. Here we present a software named SaDA for storing, retrieving and analyzing data originated from microorganism monitoring experiments. SaDA is fully integrated in the management of environmental samples, oligonucleotide sequences, microarray data and the subsequent downstream analysis procedures. It is simple and generic software, and can be extended and customized for various environmental and biomedical studies. PMID:26047146

UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Hume, Maxwell A; Barrera, Luis A; Gisselbrecht, Stephen S; Bulyk, Martha L

2015-01-01

The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k ('k-mers'). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

ChIP-chip.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions. © 2018 Cold Spring Harbor Laboratory Press.

Microarray-based determination of anti-inflammatory genes targeted by 6-(methylsulfinyl)hexyl isothiocyanate in macrophages.

PubMed

Chen, Jihua; Uto, Takuhiro; Tanigawa, Shunsuke; Yamada-Kato, Tomeo; Fujii, Makoto; Hou, DE-Xing

2010-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as 'defense, inflammatory response, cytokine activities and receptor activities' and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function.

Microarray-based determination of anti-inflammatory genes targeted by 6-(methylsulfinyl)hexyl isothiocyanate in macrophages

PubMed Central

CHEN, JIHUA; UTO, TAKUHIRO; TANIGAWA, SHUNSUKE; YAMADA-KATO, TOMEO; FUJII, MAKOTO; HOU, DE-XING

2010-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as ‘defense, inflammatory response, cytokine activities and receptor activities’ and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function. PMID:23136589

cn.FARMS: a latent variable model to detect copy number variations in microarray data with a low false discovery rate.

PubMed

Clevert, Djork-Arné; Mitterecker, Andreas; Mayr, Andreas; Klambauer, Günter; Tuefferd, Marianne; De Bondt, An; Talloen, Willem; Göhlmann, Hinrich; Hochreiter, Sepp

2011-07-01

Cost-effective oligonucleotide genotyping arrays like the Affymetrix SNP 6.0 are still the predominant technique to measure DNA copy number variations (CNVs). However, CNV detection methods for microarrays overestimate both the number and the size of CNV regions and, consequently, suffer from a high false discovery rate (FDR). A high FDR means that many CNVs are wrongly detected and therefore not associated with a disease in a clinical study, though correction for multiple testing takes them into account and thereby decreases the study's discovery power. For controlling the FDR, we propose a probabilistic latent variable model, 'cn.FARMS', which is optimized by a Bayesian maximum a posteriori approach. cn.FARMS controls the FDR through the information gain of the posterior over the prior. The prior represents the null hypothesis of copy number 2 for all samples from which the posterior can only deviate by strong and consistent signals in the data. On HapMap data, cn.FARMS clearly outperformed the two most prevalent methods with respect to sensitivity and FDR. The software cn.FARMS is publicly available as a R package at http://www.bioinf.jku.at/software/cnfarms/cnfarms.html.

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

PubMed

Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

2005-06-01

To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

SaDA: From Sampling to Data Analysis-An Extensible Open Source Infrastructure for Rapid, Robust and Automated Management and Analysis of Modern Ecological High-Throughput Microarray Data.

PubMed

Singh, Kumar Saurabh; Thual, Dominique; Spurio, Roberto; Cannata, Nicola

2015-06-03

One of the most crucial characteristics of day-to-day laboratory information management is the collection, storage and retrieval of information about research subjects and environmental or biomedical samples. An efficient link between sample data and experimental results is absolutely important for the successful outcome of a collaborative project. Currently available software solutions are largely limited to large scale, expensive commercial Laboratory Information Management Systems (LIMS). Acquiring such LIMS indeed can bring laboratory information management to a higher level, but most of the times this requires a sufficient investment of money, time and technical efforts. There is a clear need for a light weighted open source system which can easily be managed on local servers and handled by individual researchers. Here we present a software named SaDA for storing, retrieving and analyzing data originated from microorganism monitoring experiments. SaDA is fully integrated in the management of environmental samples, oligonucleotide sequences, microarray data and the subsequent downstream analysis procedures. It is simple and generic software, and can be extended and customized for various environmental and biomedical studies.

Correction of Spatial Bias in Oligonucleotide Array Data

PubMed Central

Lemieux, Sébastien

2013-01-01

Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs) for each intended target, on average, correlate with their target's true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users' current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays). A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias. PMID:23573083

Method for widespread microRNA-155 inhibition prolongs survival in ALS-model mice

PubMed Central

Koval, Erica D.; Shaner, Carey; Zhang, Peter; du Maine, Xavier; Fischer, Kimberlee; Tay, Jia; Chau, B. Nelson; Wu, Gregory F.; Miller, Timothy M.

2013-01-01

microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1)G93A rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1G93A mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS. PMID:23740943

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

PubMed

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

2006-06-12

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

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

PubMed Central

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

2018-01-01

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

Microbial Diagnostic Array Workstation (MDAW): a web server for diagnostic array data storage, sharing and analysis

PubMed Central

Scaria, Joy; Sreedharan, Aswathy; Chang, Yung-Fu

2008-01-01

Background Microarrays are becoming a very popular tool for microbial detection and diagnostics. Although these diagnostic arrays are much simpler when compared to the traditional transcriptome arrays, due to the high throughput nature of the arrays, the data analysis requirements still form a bottle neck for the widespread use of these diagnostic arrays. Hence we developed a new online data sharing and analysis environment customised for diagnostic arrays. Methods Microbial Diagnostic Array Workstation (MDAW) is a database driven application designed in MS Access and front end designed in ASP.NET. Conclusion MDAW is a new resource that is customised for the data analysis requirements for microbial diagnostic arrays. PMID:18811969

Microbial Diagnostic Array Workstation (MDAW): a web server for diagnostic array data storage, sharing and analysis.

PubMed

Scaria, Joy; Sreedharan, Aswathy; Chang, Yung-Fu

2008-09-23

Microarrays are becoming a very popular tool for microbial detection and diagnostics. Although these diagnostic arrays are much simpler when compared to the traditional transcriptome arrays, due to the high throughput nature of the arrays, the data analysis requirements still form a bottle neck for the widespread use of these diagnostic arrays. Hence we developed a new online data sharing and analysis environment customised for diagnostic arrays. Microbial Diagnostic Array Workstation (MDAW) is a database driven application designed in MS Access and front end designed in ASP.NET. MDAW is a new resource that is customised for the data analysis requirements for microbial diagnostic arrays.

NAIMA as a solution for future GMO diagnostics challenges.

PubMed

Dobnik, David; Morisset, Dany; Gruden, Kristina

2010-03-01

In the field of genetically modified organism (GMO) diagnostics, real-time PCR has been the method of choice for target detection and quantification in most laboratories. Despite its numerous advantages, however, the lack of a true multiplexing option may render real-time PCR less practical in the face of future GMO detection challenges such as the multiplicity and increasing complexity of new transgenic events, as well as the repeated occurrence of unauthorized GMOs on the market. In this context, we recently reported the development of a novel multiplex quantitative DNA-based target amplification method, named NASBA implemented microarray analysis (NAIMA), which is suitable for sensitive, specific and quantitative detection of GMOs on a microarray. In this article, the performance of NAIMA is compared with that of real-time PCR, the focus being their performances in view of the upcoming challenge to detect/quantify an increasing number of possible GMOs at a sustainable cost and affordable staff effort. Finally, we present our conclusions concerning the applicability of NAIMA for future use in GMO diagnostics.

An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase

PubMed Central

Kohlmann, Alexander; Kipps, Thomas J; Rassenti, Laura Z; Downing, James R; Shurtleff, Sheila A; Mills, Ken I; Gilkes, Amanda F; Hofmann, Wolf-Karsten; Basso, Giuseppe; Dell’Orto, Marta Campo; Foà, Robin; Chiaretti, Sabina; De Vos, John; Rauhut, Sonja; Papenhausen, Peter R; Hernández, Jesus M; Lumbreras, Eva; Yeoh, Allen E; Koay, Evelyn S; Li, Rachel; Liu, Wei-min; Williams, Paul M; Wieczorek, Lothar; Haferlach, Torsten

2008-01-01

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability. PMID:18573112

Recent Methods for Purification and Structure Determination of Oligonucleotides.

PubMed

Zhang, Qiulong; Lv, Huanhuan; Wang, Lili; Chen, Man; Li, Fangfei; Liang, Chao; Yu, Yuanyuan; Jiang, Feng; Lu, Aiping; Zhang, Ge

2016-12-18

Aptamers are single-stranded DNA or RNA oligonucleotides that can interact with target molecules through specific three-dimensional structures. The excellent features, such as high specificity and affinity for target proteins, small size, chemical stability, low immunogenicity, facile chemical synthesis, versatility in structural design and engineering, and accessible for site-specific modifications with functional moieties, make aptamers attractive molecules in the fields of clinical diagnostics and biopharmaceutical therapeutics. However, difficulties in purification and structural identification of aptamers remain a major impediment to their broad clinical application. In this mini-review, we present the recently attractive developments regarding the purification and identification of aptamers. We also discuss the advantages, limitations, and prospects for the major methods applied in purifying and identifying aptamers, which could facilitate the application of aptamers.

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

PubMed

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

2017-01-01

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

RankProd Combined with Genetic Algorithm Optimized Artificial Neural Network Establishes a Diagnostic and Prognostic Prediction Model that Revealed C1QTNF3 as a Biomarker for Prostate Cancer.

PubMed

Hou, Qi; Bing, Zhi-Tong; Hu, Cheng; Li, Mao-Yin; Yang, Ke-Hu; Mo, Zu; Xie, Xiang-Wei; Liao, Ji-Lin; Lu, Yan; Horie, Shigeo; Lou, Ming-Wu

2018-06-01

Prostate cancer (PCa) is the most commonly diagnosed cancer in males in the Western world. Although prostate-specific antigen (PSA) has been widely used as a biomarker for PCa diagnosis, its results can be controversial. Therefore, new biomarkers are needed to enhance the clinical management of PCa. From publicly available microarray data, differentially expressed genes (DEGs) were identified by meta-analysis with RankProd. Genetic algorithm optimized artificial neural network (GA-ANN) was introduced to establish a diagnostic prediction model and to filter candidate genes. The diagnostic and prognostic capability of the prediction model and candidate genes were investigated in both GEO and TCGA datasets. Candidate genes were further validated by qPCR, Western Blot and Tissue microarray. By RankProd meta-analyses, 2306 significantly up- and 1311 down-regulated probes were found in 133 cases and 30 controls microarray data. The overall accuracy rate of the PCa diagnostic prediction model, consisting of a 15-gene signature, reached up to 100% in both the training and test dataset. The prediction model also showed good results for the diagnosis (AUC = 0.953) and prognosis (AUC of 5 years overall survival time = 0.808) of PCa in the TCGA database. The expression levels of three genes, FABP5, C1QTNF3 and LPHN3, were validated by qPCR. C1QTNF3 high expression was further validated in PCa tissue by Western Blot and Tissue microarray. In the GEO datasets, C1QTNF3 was a good predictor for the diagnosis of PCa (GSE6956: AUC = 0.791; GSE8218: AUC = 0.868; GSE26910: AUC = 0.972). In the TCGA database, C1QTNF3 was significantly associated with PCa patient recurrence free survival (P < .001, AUC = 0.57). In this study, we have developed a diagnostic and prognostic prediction model for PCa. C1QTNF3 was revealed as a promising biomarker for PCa. This approach can be applied to other high-throughput data from different platforms for the discovery of oncogenes or biomarkers in different kinds of diseases. Copyright © 2018. Published by Elsevier B.V.

Genome Expression Pathway Analysis Tool – Analysis and visualization of microarray gene expression data under genomic, proteomic and metabolic context

PubMed Central

Weniger, Markus; Engelmann, Julia C; Schultz, Jörg

2007-01-01

Background Regulation of gene expression is relevant to many areas of biology and medicine, in the study of treatments, diseases, and developmental stages. Microarrays can be used to measure the expression level of thousands of mRNAs at the same time, allowing insight into or comparison of different cellular conditions. The data derived out of microarray experiments is highly dimensional and often noisy, and interpretation of the results can get intricate. Although programs for the statistical analysis of microarray data exist, most of them lack an integration of analysis results and biological interpretation. Results We have developed GEPAT, Genome Expression Pathway Analysis Tool, offering an analysis of gene expression data under genomic, proteomic and metabolic context. We provide an integration of statistical methods for data import and data analysis together with a biological interpretation for subsets of probes or single probes on the chip. GEPAT imports various types of oligonucleotide and cDNA array data formats. Different normalization methods can be applied to the data, afterwards data annotation is performed. After import, GEPAT offers various statistical data analysis methods, as hierarchical, k-means and PCA clustering, a linear model based t-test or chromosomal profile comparison. The results of the analysis can be interpreted by enrichment of biological terms, pathway analysis or interaction networks. Different biological databases are included, to give various information for each probe on the chip. GEPAT offers no linear work flow, but allows the usage of any subset of probes and samples as a start for a new data analysis. GEPAT relies on established data analysis packages, offers a modular approach for an easy extension, and can be run on a computer grid to allow a large number of users. It is freely available under the LGPL open source license for academic and commercial users at . Conclusion GEPAT is a modular, scalable and professional-grade software integrating analysis and interpretation of microarray gene expression data. An installation available for academic users can be found at . PMID:17543125

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

PubMed Central

Galfalvy, Hanga C; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Pavlidis, Paul; Ellis, Steven P; Mann, J John; Sibille, Etienne; Arango, Victoria

2003-01-01

Background Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Results Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. Conclusion In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects. PMID:12962547

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

PubMed

Galfalvy, Hanga C; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Pavlidis, Paul; Ellis, Steven P; Mann, J John; Sibille, Etienne; Arango, Victoria

2003-09-08

Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods. Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression. In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects.

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

PubMed Central

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

2013-01-01

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

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

PubMed Central

Kamata, Teddy; Natesan, Mohan; Warfield, Kelly; Aman, M. Javad

2014-01-01

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. PMID:25230936

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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A bioinformatics approach to identify patients with symptomatic peanut allergy using peptide microarray immunoassay

PubMed Central

Lin, Jing; Bruni, Francesca M.; Fu, Zhiyan; Maloney, Jennifer; Bardina, Ludmilla; Boner, Attilio L.; Gimenez, Gustavo; Sampson, Hugh A.

2013-01-01

Background Peanut allergy is relatively common, typically permanent, and often severe. Double-blind, placebo-controlled food challenge is considered the gold standard for the diagnosis of food allergy–related disorders. However, the complexity and potential of double-blind, placebo-controlled food challenge to cause life-threatening allergic reactions affects its clinical application. A laboratory test that could accurately diagnose symptomatic peanut allergy would greatly facilitate clinical practice. Objective We sought to develop an allergy diagnostic method that could correctly predict symptomatic peanut allergy by using peptide microarray immunoassays and bioinformatic methods. Methods Microarray immunoassays were performed by using the sera from 62 patients (31 with symptomatic peanut allergy and 31 who had outgrown their peanut allergy or were sensitized but were clinically tolerant to peanut). Specific IgE and IgG4 binding to 419 overlapping peptides (15 mers, 3 offset) covering the amino acid sequences of Ara h 1, Ara h 2, and Ara h 3 were measured by using a peptide microarray immunoassay. Bioinformatic methods were applied for data analysis. Results Individuals with peanut allergy showed significantly greater IgE binding and broader epitope diversity than did peanut-tolerant individuals. No significant difference in IgG4 binding was found between groups. By using machine learning methods, 4 peptide biomarkers were identified and prediction models that can predict the outcome of double-blind, placebo-controlled food challenges with high accuracy were developed by using a combination of the biomarkers. Conclusions In this study, we developed a novel diagnostic approach that can predict peanut allergy with high accuracy by combining the results of a peptide microarray immunoassay and bioinformatic methods. Further studies are needed to validate the efficacy of this assay in clinical practice. PMID:22444503

Characterization of two familial cases presenting with a syndromic specific learning disorder and carrying (17q;21q) unbalanced translocations.

PubMed

Coton, Julie; Labalme, Audrey; Till, Marianne; Bussy, Gerald; Krifi Papoz, Sonia; Lesca, Gaetan; Heron, Delphine; Sanlaville, Damien; Edery, Patrick; des Portes, Vincent; Rossi, Massimiliano

2018-05-01

Chromosomal microarray (CMA) can detect pathogenic copy number variations in 15-20% of individuals with intellectual disability and in 10% of patients with autism spectrum disorders. The diagnostic rate in specific learning disorders (SLD) is unknown. Our study emphasizes the usefulness of CMA in the diagnostic workout assessment of familial SLD.

[Chromosome banding analysis of peripheral blood lymphocytes stimulated with IL2 and CpG oligonucleotide DSP30 in patients with chronic lymphocytic leukemia].

PubMed

Stěpanovská, K; Vaňková, G; Némethová, V; Tomášiková, L; Smuhařová, P; Divíšková, E; Vallová, V; Kuglík, P; Plevová, K; Oltová, A; Doubek, M; Pospíšilová, S; Mayer, J

2013-01-01

Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics. In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13. Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis. Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

PubMed

Jara-Acevedo, Maria; Teodosio, Cristina; Sanchez-Muñoz, Laura; Álvarez-Twose, Ivan; Mayado, Andrea; Caldas, Carolina; Matito, Almudena; Morgado, José M; Muñoz-González, Javier I; Escribano, Luis; Garcia-Montero, Andrés C; Orfao, Alberto

2015-08-01

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Gene expression profiling of Listeria monocytogenes strain F2365 during growth in ultrahigh-temperature-processed skim milk.

PubMed

Liu, Yanhong; Ream, Amy

2008-11-01

To study how Listeria monocytogenes survives and grows in ultrahigh-temperature-processed (UHT) skim milk, microarray technology was used to monitor the gene expression profiles of strain F2365 in UHT skim milk. Total RNA was isolated from strain F2365 in UHT skim milk after 24 h of growth at 4 degrees C, labeled with fluorescent dyes, and hybridized to "custom-made" commercial oligonucleotide (35-mers) microarray chips containing the whole genome of L. monocytogenes strain F2365. Compared to L. monocytogenes grown in brain heart infusion (BHI) broth for 24 h at 4 degrees C, 26 genes were upregulated (more-than-twofold increase) in UHT skim milk, whereas 14 genes were downregulated (less-than-twofold decrease). The upregulated genes included genes encoding transport and binding proteins, transcriptional regulators, proteins in amino acid biosynthesis and energy metabolism, protein synthesis, cell division, and hypothetical proteins. The downregulated genes included genes that encode transport and binding proteins, protein synthesis, cellular processes, cell envelope, energy metabolism, a transcriptional regulator, and an unknown protein. The gene expression changes determined by microarray assays were confirmed by real-time reverse transcriptase PCR analyses. Furthermore, cells grown in UHT skim milk displayed the same sensitivity to hydrogen peroxide as cells grown in BHI, demonstrating that the elevated levels of expression of genes encoding manganese transporter complexes in UHT skim milk did not result in changes in the oxidative stress sensitivity. To our knowledge, this report represents a novel study of global transcriptional gene expression profiling of L. monocytogenes in a liquid food.

Protein profiles associated with survival in lung adenocarcinoma

PubMed Central

Chen, Guoan; Gharib, Tarek G; Wang, Hong; Huang, Chiang-Ching; Kuick, Rork; Thomas, Dafydd G.; Shedden, Kerby A.; Misek, David E.; Taylor, Jeremy M. G.; Giordano, Thomas J.; Kardia, Sharon L. R.; Iannettoni, Mark D.; Yee, John; Hogg, Philip J.; Orringer, Mark B.; Hanash, Samir M.; Beer, David G.

2003-01-01

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer. PMID:14573703

Decreased triadin and increased calstabin2 expression in Great Danes with dilated cardiomyopathy.

PubMed

Oyama, M A; Chittur, S V; Reynolds, C A

2009-01-01

Dilated cardiomyopathy (DCM) is a common cardiac disease of Great Dane dogs, yet very little is known about the underlying molecular abnormalities that contribute to disease. Discover a set of genes that are differentially expressed in Great Dane dogs with DCM as a way to identify candidate genes for further study as well as to better understand the molecular abnormalities that underlie the disease. Three Great Dane dogs with end-stage DCM and 3 large breed control dogs. Prospective study. Transcriptional activity of 42,869 canine DNA sequences was determined with a canine-specific oligonucleotide microarray. Genome expression patterns of left ventricular tissue samples from affected Great Dane dogs were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with expression from large breed dogs with noncardiac disease. Three hundred and twenty-three transcripts were differentially expressed (> or = 2-fold change). The transcript with the greatest degree of upregulation (+61.3-fold) was calstabin2 (FKBP12.6), whereas the transcript with the greatest degree of downregulation (-9.07-fold) was triadin. Calstabin2 and triadin are both regulatory components of the cardiac ryanodine receptor (RyR2) and are critical to normal intracellular Ca2+ release and excitation-contraction coupling. Great Dane dogs with DCM demonstrate abnormal calstabin2 and triadin expression. These changes likely affect Ca2+ flux within cardiac cells and may contribute to the pathophysiology of disease. Microarray-based analysis identifies calstabin2, triadin, and RyR2 function as targets of future study.

Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice.

PubMed

Joseph, Ajay; Lee, Taewon; Moland, Carrie L; Branham, William S; Fuscoe, James C; Leakey, Julian E A; Allaben, William T; Lewis, Sherry M; Ali, Akhtar A; Desai, Varsha G

2009-04-01

Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F(1) female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I-IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.

A bimetallic nanocomposite modified genosensor for recognition and determination of thalassemia gene.

PubMed

Hamidi-Asl, Ezat; Raoof, Jahan Bakhsh; Naghizadeh, Nahid; Akhavan-Niaki, Haleh; Ojani, Reza; Banihashemi, Ali

2016-10-01

The main roles of DNA in the cells are to maintain and properly express genetic information. It is important to have analytical methods capable of fast and sensitive detection of DNA damage. DNA hybridization sensors are well suited for diagnostics and other purposes, including determination of bacteria and viruses. Beta thalassemias (βth) are due to mutations in the β-globin gene. In this study, an electrochemical biosensor which detects the sequences related to the β-globin gene issued from real samples amplified by polymerase chain reaction (PCR) is described for the first time. The biosensor relies on the immobilization of 20-mer single stranded oligonucleotide (probe) related to βth sequence on the carbon paste electrode (CPE) modified by 15% silver (Ag) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode and hybridization of this oligonucleotide with its complementary sequence (target). The extent of hybridization between the probe and target sequences was shown by using linear sweep voltammetry (LSV) with methylene blue (MB) as hybridization indicator. The selectivity of sensor was investigated using PCR samples containing non-complementary oligonucleotides. The detection limit of biosensor was calculated about 470.0pg/μL. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel in silico technology in combination with microarrays: a state-of-the-art technology for allergy diagnosis and management?

PubMed

Melioli, Giovanni; Passalacqua, Giovanni; Canonica, Giorgio W

2014-12-01

'Allergen microarrays, in poly-sensitized allergic patients, represent a real value added in the accurate IgE profiling and in the identification of allergen(s) to administer for an effective allergen immunotherapy.' Allergen microarrays (AMA) were developed in the early 2000s to improve the diagnostic pathway of patients with allergic reactions. Nowadays, AMA are constituted by more than 100 different components (either purified or recombinant), representing genuine and cross-reacting molecules from plants and animals. The cost of the procedure had suggested its use as third-level diagnostics (following in vivo- and in vitro-specific IgE tests) in poly-sensitized patients. The complexity of the interpretation had inspired the development of in silico technologies to help clinicians in their work. Both machine learning techniques and expert systems are now available. In particular, an expert system that has been recently developed not only identifies positive and negative components but also lists dangerous components and classifies patients based on their potential responsiveness to allergen immunotherapy, on the basis of published algorithms. For these characteristics, AMA represents the state-of-the-art technology for allergy diagnosis in poly-sensitized patients.

Five omic technologies are concordant in differentiating the biochemical characteristics of the berries of five grapevine (Vitis vinifera L.) cultivars.

PubMed

Ghan, Ryan; Van Sluyter, Steven C; Hochberg, Uri; Degu, Asfaw; Hopper, Daniel W; Tillet, Richard L; Schlauch, Karen A; Haynes, Paul A; Fait, Aaron; Cramer, Grant R

2015-11-16

Grape cultivars and wines are distinguishable by their color, flavor and aroma profiles. Omic analyses (transcripts, proteins and metabolites) are powerful tools for assessing biochemical differences in biological systems. Berry skins of red- (Cabernet Sauvignon, Merlot, Pinot Noir) and white-skinned (Chardonnay, Semillon) wine grapes were harvested near optimum maturity (°Brix-to-titratable acidity ratio) from the same experimental vineyard. The cultivars were exposed to a mild, seasonal water-deficit treatment from fruit set until harvest in 2011. Identical sample aliquots were analyzed for transcripts by grapevine whole-genome oligonucleotide microarray and RNAseq technologies, proteins by nano-liquid chromatography-mass spectroscopy, and metabolites by gas chromatography-mass spectroscopy and liquid chromatography-mass spectroscopy. Principal components analysis of each of five Omic technologies showed similar results across cultivars in all Omic datasets. Comparison of the processed data of genes mapped in RNAseq and microarray data revealed a strong Pearson's correlation (0.80). The exclusion of probesets associated with genes with potential for cross-hybridization on the microarray improved the correlation to 0.93. The overall concordance of protein with transcript data was low with a Pearson's correlation of 0.27 and 0.24 for the RNAseq and microarray data, respectively. Integration of metabolite with protein and transcript data produced an expected model of phenylpropanoid biosynthesis, which distinguished red from white grapes, yet provided detail of individual cultivar differences. The mild water deficit treatment did not significantly alter the abundance of proteins or metabolites measured in the five cultivars, but did have a small effect on gene expression. The five Omic technologies were consistent in distinguishing cultivar variation. There was high concordance between transcriptomic technologies, but generally protein abundance did not correlate well with transcript abundance. The integration of multiple high-throughput Omic datasets revealed complex biochemical variation amongst five cultivars of an ancient and economically important crop species.

Nucleic acids--genes, drugs, molecular lego and more.

PubMed

Häner, Robert

2010-01-01

Chemically modified nucleic acids find widespread use as tools in research, as diagnostic reagents and even as pharmaceutical compounds. On the background of antisense research and development, the synthesis and evaluation of modified oligonucleotides was intensively pursued in the early to mid nineties in corporate research of former Ciba. Most of these efforts concentrated on the development of sugar and/or backbone-modified derivatives for pharmaceutical applications. Additionally, oligonucleotide metal conjugates were investigated with the goal to develop artificial ribonucleases. Since the turn of the millennium also the potential of non-nucleosidic and non-hydrogen bonding building blocks has increasingly been recognized. Such derivatives possess unique properties that may have an impact in the fields of materials and genetic research. In this brief account, we take a personal look back on some past as well as some recent results.

Plasmodium Genus Assay Transition to the Joint Biological Agent Identification and Diagnostic System (JBAIDS)

DTIC Science & Technology

2012-07-12

readily transferable to diverse real-time PCR instrumentation. These assays are used regularly for vector surveillance and are the primary...instrumentation ( FilmArray ) is under assessment. Assay oligonucleotide sequences and formulations are available for use in future joint projects...Plasmodium real-time PCR detection capability has been challenging. During 2006, the Division of Entomology, WRAIR designed and developed a

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

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

PubMed

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

2008-07-01

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

Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation.

PubMed

Kulterer, Birgit; Friedl, Gerald; Jandrositz, Anita; Sanchez-Cabo, Fatima; Prokesch, Andreas; Paar, Christine; Scheideler, Marcel; Windhager, Reinhard; Preisegger, Karl-Heinz; Trajanoski, Zlatko

2007-03-12

Human mesenchymal stem cells (MSC) with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts. The results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-beta2 and BMPs. With a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.

Gene expression profiling of mucolipidosis type IV fibroblasts reveals deregulation of genes with relevant functions in lysosome physiology.

PubMed

Bozzato, Andrea; Barlati, Sergio; Borsani, Giuseppe

2008-04-01

Mucolipidosis type IV (MLIV, MIM 252650) is an autosomal recessive lysosomal storage disorder that causes mental and motor retardation as well as visual impairment. The lysosomal storage defect in MLIV is consistent with abnormalities of membrane traffic and organelle dynamics in the late endocytic pathway. MLIV is caused by mutations in the MCOLN1 gene, which codes for mucolipin-1 (MLN1), a member of the large family of transient receptor potential (TRP) cation channels. Although a number of studies have been performed on mucolipin-1, the pathological mechanisms underlying MLIV are not fully understood. To identify genes that characterize pathogenic changes in mucolipidosis type IV, we compared the expression profiles of three MLIV and three normal skin fibroblasts cell lines using oligonucleotide microarrays. Genes that were differentially expressed in patients' cells were identified. 231 genes were up-regulated, and 116 down-regulated. Real-Time RT-PCR performed on selected genes in six independent MLIV fibroblasts cell lines was generally consistent with the microarray findings. This study allowed to evidence the modulation at the transcriptional level of a discrete number of genes relevant in biological processes which are altered in the disease such as endosome/lysosome trafficking, lysosome biogenesis, organelle acidification and lipid metabolism.

Successful Application of Microarray Technology to Microdissected Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

Coudry, Renata A.; Meireles, Sibele I.; Stoyanova, Radka; Cooper, Harry S.; Carpino, Alan; Wang, Xianqun; Engstrom, Paul F.; Clapper, Margie L.

2007-01-01

The establishment of a reliable method for using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue would provide an opportunity to obtain novel gene expression data from the vast amounts of archived tissue. A custom-designed 22,000 oligonucleotide array was used in the present study to compare the gene expression profile of colonic epithelial cells isolated by laser capture microdissection from FFPE-archived samples with that of the same cell population from matched frozen samples, the preferred source of RNA. Total RNA was extracted from FFPE tissues, amplified, and labeled using the Paradise Reagent System. The quality of the input RNA was assessed by the Bioanalyzer profile, reverse transcriptase-polymerase chain reaction, and agarose gel electrophoresis. The results demonstrate that it is possible to obtain reliable microarray data from FFPE samples using RNA acquired by laser capture microdissection. The concordance between matched FFPE and frozen samples was evaluated and expressed as a Pearson’s correlation coefficient, with values ranging from 0.80 to 0.97. The presence of ribosomal RNA peaks in FFPE-derived RNA was reflected by a high correlation with paired frozen samples. A set of practical recommendations for evaluating the RNA integrity and quality in FFPE samples is reported. PMID:17251338

A new normalizing algorithm for BAC CGH arrays with quality control metrics.

PubMed

Miecznikowski, Jeffrey C; Gaile, Daniel P; Liu, Song; Shepherd, Lori; Nowak, Norma

2011-01-01

The main focus in pin-tip (or print-tip) microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH) experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose "SmoothArray", a new method to preprocess comparative genomic hybridization (CGH) bacterial artificial chromosome (BAC) arrays and we show the effects on a cancer dataset. As part of our R software package "aCGHplus," this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology.

Genomic expression patterns of cardiac tissues from dogs with dilated cardiomyopathy.

PubMed

Oyama, Mark A; Chittur, Sridar

2005-07-01

To evaluate global genome expression patterns of left ventricular tissues from dogs with dilated cardiomyopathy (DCM). Tissues obtained from the left ventricle of 2 Doberman Pinschers with end-stage DCM and 5 healthy control dogs. Transcriptional activities of 23,851 canine DNA sequences were determined by use of an oligonucleotide microarray. Genome expression patterns of DCM tissue were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with gene expression for tissues from 5 healthy control dogs. 478 transcripts were differentially expressed (> or = 2.5-fold change). In DCM tissue, expression of 173 transcripts was upregulated and expression of 305 transcripts was downregulated, compared with expression for control tissues. Of the 478 transcripts, 167 genes could be specifically identified. These genes were grouped into 1 of 8 categories on the basis of their primary physiologic function. Grouping revealed that pathways involving cellular energy production, signaling and communication, and cell structure were generally downregulated, whereas pathways involving cellular defense and stress responses were upregulated. Many previously unreported genes that may contribute to the pathophysiologic aspects of heart disease were identified. Evaluation of global expression patterns provides a molecular portrait of heart failure, yields insights into the pathophysiologic aspects of DCM, and identifies intriguing genes and pathways for further study.

DNA Microarray Profiling Highlights Nrf2-Mediated Chemoprevention Targeted by Wasabi-Derived Isothiocyanates in HepG2 Cells.

PubMed

Trio, Phoebe Zapanta; Kawahara, Atsuyoshi; Tanigawa, Shunsuke; Sakao, Kozue; Hou, De-Xing

2017-01-01

6-MSITC and 6-MTITC are sulforaphane (SFN) analogs found in Japanese Wasabi. As we reported previously, Wasabi isothiocyanates (ITCs) are activators of Nrf2-antioxidant response element pathway, and also inhibitors of pro-inflammatory cyclooxygenase-2. This study is the first to assess the global changes in transcript levels by Wasabi ITCs, comparing with SFN, in HepG2 cells. We performed comparative gene expression profiling by treating HepG2 cells with ITCs, followed by DNA microarray analyses using HG-U133 plus 2.0 oligonucleotide array. Partial array data on selected gene products were confirmed by RT-PCR and Western blotting. Ingenuity Pathway Analysis (IPA) was used to identify functional subsets of genes and biologically significant network pathways. 6-MTITC showed the highest number of differentially altered (≥2 folds) gene expression, of which 114 genes were upregulated and 75 were downregulated. IPA revealed that Nrf2-mediated pathway, together with glutamate metabolism, is the common significantly modulated pathway across treatments. Interestingly, 6-MSITC exhibited the most potent effect toward Nrf2-mediated pathway. Our data suggest that 6-MSITC could exert chemopreventive role against cancer through its underlying antioxidant activity via the activation of Nrf2-mediated subsequent induction of cytoprotective genes.

Increased expression of protein kinase A inhibitor alpha (PKI-alpha) and decreased PKA-regulated genes in chronic intermittent alcohol exposure.

PubMed

Repunte-Canonigo, Vez; Lutjens, Robert; van der Stap, Lena D; Sanna, Pietro Paolo

2007-03-23

Intermittent models of alcohol exposure that mimic human patterns of alcohol consumption produce profound physiological and biochemical changes and induce rapid increases in alcohol self-administration. We used high-density oligonucleotide microarrays to investigate gene expression changes during chronic intermittent alcohol exposure in three brain regions that receive mesocorticolimbic dopaminergic projections and that are believed to be involved in alcohol's reinforcing actions: the medial prefrontal cortex, the nucleus accumbens and the amygdala. An independent replication of the experiment was used for RT-PCR validation of the microarray results. The protein kinase A inhibitor alpha (PKI-alpha, Pkia), a member of the endogenous PKI family implicated in reducing nuclear PKA activity, was found to be increased in all three regions tested. Conversely, we observed a downregulation of the expression of several PKA-regulated transcripts in one or more of the brain regions studied, including the activity and neurotransmitter-regulated early gene (Ania) - 1, -3, -7, -8, the transcription factors Egr1 and NGFI-B (Nr4a1) and the neuropeptide NPY. Reduced expression of PKA-regulated genes in mesocorticolimbic projection areas may have motivational significance in the rapid increase in alcohol self-administration induced by intermittent alcohol exposure.

Robust diagnosis of non-Hodgkin lymphoma phenotypes validated on gene expression data from different laboratories.

PubMed

Bhanot, Gyan; Alexe, Gabriela; Levine, Arnold J; Stolovitzky, Gustavo

2005-01-01

A major challenge in cancer diagnosis from microarray data is the need for robust, accurate, classification models which are independent of the analysis techniques used and can combine data from different laboratories. We propose such a classification scheme originally developed for phenotype identification from mass spectrometry data. The method uses a robust multivariate gene selection procedure and combines the results of several machine learning tools trained on raw and pattern data to produce an accurate meta-classifier. We illustrate and validate our method by applying it to gene expression datasets: the oligonucleotide HuGeneFL microarray dataset of Shipp et al. (www.genome.wi.mit.du/MPR/lymphoma) and the Hu95Av2 Affymetrix dataset (DallaFavera's laboratory, Columbia University). Our pattern-based meta-classification technique achieves higher predictive accuracies than each of the individual classifiers , is robust against data perturbations and provides subsets of related predictive genes. Our techniques predict that combinations of some genes in the p53 pathway are highly predictive of phenotype. In particular, we find that in 80% of DLBCL cases the mRNA level of at least one of the three genes p53, PLK1 and CDK2 is elevated, while in 80% of FL cases, the mRNA level of at most one of them is elevated.

The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia.

PubMed

Alyamani, Essam J; Khiyami, Anamil M; Booq, Rayan Y; Majrashi, Majed A; Bahwerth, Fayez S; Rechkina, Elena

2017-01-06

The infection and prevalence of extended-spectrum β-lactamases (ESBLs) is a worldwide problem, and the presence of ESBLs varies between countries. In this study, we investigated the occurrence of plasmid-mediated ESBL/AmpC/carbapenemase/aminoglycoside resistance gene expression in Escherichia coli using phenotypic and genotypic techniques. A total of 58 E. coli isolates were collected from hospitals in the city of Makkah and screened for the production of ESBL/AmpC/carbapenemase/aminoglycoside resistance genes. All samples were subjected to phenotypic and genotypic analyses. The antibiotic susceptibility of the E. coli isolates was determined using the Vitek-2 system and the minimum inhibitory concentration (MIC) assay. Antimicrobial agents tested using the Vitek 2 system and MIC assay included the expanded-spectrum (or third-generation) cephalosporins (e.g., cefoxitin, cefepime, aztreonam, cefotaxime, ceftriaxone, and ceftazidime) and carbapenems (meropenem and imipenem). Reported positive isolates were investigated using genotyping technology (oligonucleotide microarray-based assay and PCR). The genotyping investigation was focused on ESBL variants and the AmpC, carbapenemase and aminoglycoside resistance genes. E. coli was phylogenetically grouped, and the clonality of the isolates was studied using multilocus sequence typing (MLST). Our E. coli isolates exhibited different levels of resistance to ESBL drugs, including ampicillin (96.61%), cefoxitin (15.25%), ciprofloxacin (79.66%), cefepime (75.58%), aztreonam (89.83%), cefotaxime (76.27%), ceftazidime (81.36%), meropenem (0%) and imipenem (0%). Furthermore, the distribution of ESBL-producing E. coli was consistent with the data obtained using an oligonucleotide microarray-based assay and PCR genotyping against genes associated with β-lactam resistance. ST131 was the dominant sequence type lineage of the isolates and was the most uropathogenic E. coli lineage. The E. coli isolates also carried aminoglycoside resistance genes. The evolution and prevalence of ESBL-producing E. coli may be rapidly accelerating in Saudi Arabia due to the high visitation seasons (especially to the city of Makkah). The health authority in Saudi Arabia should monitor the level of drug resistance in all general hospitals to reduce the increasing trend of microbial drug resistance and the impact on patient therapy.

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

PubMed Central

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian; Ulrich, Eldon L.; Zhao, Qin; Wrobel, Russell L.; Newman, Craig S.; Fox, Brian G.; Phillips, George N.; Markley, John L.; Sussman, Michael R.

2005-01-01

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron–exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions “antisense” to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage. PMID:15755812

Salivary gland carcinosarcoma: oligonucleotide array CGH reveals similar genomic profiles in epithelial and mesenchymal components.

PubMed

Vékony, Hedy; Leemans, C René; Ylstra, Bauke; Meijer, Gerrit A; van der Waal, Isaäc; Bloemena, Elisabeth

2009-03-01

In this study, we present a case of parotid gland de novo carcinosarcoma. Salivary gland carcinosarcoma (or true malignant mixed tumor) is a rare biphasic neoplasm, composed of both malignant epithelial and malignant mesenchymal components. It is yet unclear whether these two phenotypes occur by collision of two independent tumors or if they are of clonal origin. To analyze the clonality of the different morphologic tumor components, oligonucleotide microarray-based comparative genomic hybridization (oaCGH) was performed on the carcinoma and the sarcoma entity separately. This technique enables a high-resolution, genome-wide overview of the chromosomal alterations in the distinct tumor elements. Analysis of both fractions showed a high number of DNA copy number changes. Losses were more prevalent than gains (82 and 49, respectively). The carcinomatous element displayed more chromosomal aberrations than the sarcomatous component. Specific amplifications of MUC20 (in mesenchymal element) and BMI-1 (in both elements) loci were observed. Overall homology between the two genomic profiles was 75%. DNA copy number profiles of the epithelial and mesenchymal components in this salivary gland carcinosarcoma displayed extensive overlap, indicating a monoclonal origin. Since losses are shared to a larger extent than gains, they seem to be more essential for initial oncogenic events. Furthermore, specific amplifications of a mucin and a Polycomb group gene imply these proteins in the tumorigenesis of carcinosarcomas.

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian;

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

PubMed Central

van Huet, Ramon A. C.; Pierrache, Laurence H.M.; Meester-Smoor, Magda A.; Klaver, Caroline C.W.; van den Born, L. Ingeborgh; Hoyng, Carel B.; de Wijs, Ilse J.; Collin, Rob W. J.; Hoefsloot, Lies H.

2015-01-01

Purpose To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon–intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis. PMID:25999674

Chromosomal microarray analysis in developmental delay and intellectual disability with comorbid conditions.

PubMed

Fan, Yanjie; Wu, Yanming; Wang, Lili; Wang, Yu; Gong, Zhuwen; Qiu, Wenjuan; Wang, Jingmin; Zhang, Huiwen; Ji, Xing; Ye, Jun; Han, Lianshu; Jin, Xingming; Shen, Yongnian; Li, Fei; Xiao, Bing; Liang, Lili; Zhang, Xia; Liu, Xiaomin; Gu, Xuefan; Yu, Yongguo

2018-05-24

Developmental delay (DD) and intellectual disability (ID) are frequently associated with a broad spectrum of additional phenotypes. Chromosomal microarray analysis (CMA) has been recommended as a first-tier test for DD/ID in general, whereas the diagnostic yield differs significantly among DD/ID patients with different comorbid conditions. To investigate the genotype-phenotype correlation, we examined the characteristics of identified pathogenic copy number variations (pCNVs) and compared the diagnostic yields among patient subgroups with different co-occurring conditions. This study is a retrospective review of CMA results generated from a mixed cohort of 710 Chinese patients with DD/ID. A total of 247 pCNVs were identified in 201 patients (28%). A large portion of these pCNVs were copy number losses, and the size of copy number losses was generally smaller than gains. The diagnostic yields were significantly higher in subgroups with co-occurring congenital heart defects (55%), facial dysmorphism (39%), microcephaly (34%) or hypotonia (35%), whereas co-occurring conditions of skeletal malformation (26%), brain malformation (24%) or epilepsy (24%) did not alter the yield. In addition, the diagnostic yield nominally correlated with ID severity. Varied yields exist in DD/ID patients with different phenotypic presentation. The presence of comorbid conditions can be among factors to consider when planning CMA.

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

PubMed

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

2014-06-17

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

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

NASA Astrophysics Data System (ADS)

Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

2006-01-01

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide

NASA Astrophysics Data System (ADS)

Rull, Jordi; Nonglaton, Guillaume; Costa, Guillaume; Fontelaye, Caroline; Marchi-Delapierre, Caroline; Ménage, Stéphane; Marchand, Gilles

2015-11-01

The functionalization of silicon oxide based substrates using silanes is generally performed through liquid phase methodologies. These processes involve a huge quantity of potentially toxic solvents and present some important disadvantages for the functionalization of microdevices or porous materials, for example the low diffusion. To overcome this drawback, solvent-free methodologies like molecular vapor deposition (MVD) or supercritical fluid deposition (SFD) have been developed. In this paper, the deposition process of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon oxide using supercritical carbon dioxide (scCO2) as a solvent is studied for the first time. The oxirane ring of epoxy silanes readily reacts with amine group and is of particular interest for the grafting of amino-modified oligonucleotides or antibodies for diagnostic application. Then the ability of this specific EBTMOS layer to react with amine functions has been evaluated using the immobilization of amino-modified oligonucleotide probes. The presence of the probes is revealed by fluorescence using hybridization with a fluorescent target oligonucleotide. The performances of SFD of EBTMOS have been optimized and then compared with the dip coating and molecular vapor deposition methods, evidencing a better grafting efficiency and homogeneity, a lower reaction time in addition to the eco-friendly properties of the supercritical carbon dioxide. The epoxysilane layers have been characterized by surface enhanced ellipsometric contrast optical technique, atomic force microscopy, multiple internal reflection infrared spectroscopy and X-ray photoelectron spectroscopy. The shelf life of the 3,4-epoxybutyltrimethoxysilane coating layer has also been studied. Finally, two different strategies of NH2-oligonucleotide grafting on EBTMOS coating layer have been compared, i.e. reductive amination and nucleophilic substitution, SN2. This EBTMOS based coating layer can be used for a wide range of applications such as the preparation of new supported and recoverable catalysts and new integrated silicon microdevices for healthcare purposes.

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

PubMed

Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

Detection of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia in primary endodontic infections in a Chinese population.

PubMed

Cao, H; Qi, Z; Jiang, H; Zhao, J; Liu, Z; Tang, Z

2012-08-01

To assess the prevalence of three black-pigmented bacterial species (Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia) using microarray technology in root canals of teeth associated with primary endodontic infections in a Chinese population. Microbial samples were taken from root canals of 80 teeth with pulp necrosis and primary endodontic infections in a Chinese population. DNA extracted from the samples was amplified by PCR with universal bacterial primers based on 16S rRNA gene sequences, and the products hybridized with the microarrays in which the specific oligonucleotide probes were added. The results of hybridization were screened by a confocal laser scanner. Pearson chi-square test and the two-sided Fisher exact test were used to analyse whether a significant association existed between the species and symptoms as well as in co-existence of two target organisms by a statistical software package (SAS 8.02). The 16S rRNA gene microarray detected at least one of the three test species in 76% of the study teeth. P. endodontalis, P. gingivalis and P. intermedia were found in 50%, 33% and 45%, respectively. A significant association was found in the presence of the pair P. endodontalis / P. gingivalis (P < 0.005). Both P. endodontalis (P <0.05) and P. gingivalis (P <0.005) had a statistically significant association with the presence of a sinus tract. The simultaneous presence of P. endodontalis and P. gingivalis was also associated with the presence of a sinus tract (P<0.005) and abscess formation (P<0.05). The three black-pigmented bacteria were prevalent in teeth with pulp necrosis and primary endodontic infections in a Chinese population. P. gingivalis and P. endodontalis were associated with the presence of sinus tract and abscess formation. © 2012 International Endodontic Journal.

Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes

PubMed Central

Pappas, Christopher T.; Sram, Jakub; Moskvin, Oleg V.; Ivanov, Pavel S.; Mackenzie, R. Christopher; Choudhary, Madhusudan; Land, Miriam L.; Larimer, Frank W.; Kaplan, Samuel; Gomelsky, Mark

2004-01-01

A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes—aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis—were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium. PMID:15231807

Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

PubMed

Villeneuve, L; Wang, Rong-Lin; Bencic, David C; Biales, Adam D; Martinović, Dalma; Lazorchak, James M; Toth, Gregory; Ankley, Gerald T

2009-08-01

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

The prevalence of chromosomal deletions relating to developmental delay and/or intellectual disability in human euploid blastocysts.

PubMed

He, Wenyin; Sun, Xiaofang; Liu, Lian; Li, Man; Jin, Hua; Wang, Wei-Hua

2014-01-01

Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34-149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision.

Non-Small-Cell Lung Cancer Molecular Signatures Recapitulate Lung Developmental Pathways

PubMed Central

Borczuk, Alain C.; Gorenstein, Lyall; Walter, Kristin L.; Assaad, Adel A.; Wang, Liqun; Powell, Charles A.

2003-01-01

Current paradigms hold that lung carcinomas arise from pleuripotent stem cells capable of differentiation into one or several histological types. These paradigms suggest lung tumor cell ontogeny is determined by consequences of gene expression that recapitulate events important in embryonic lung development. Using oligonucleotide microarrays, we acquired gene profiles from 32 microdissected non-small-cell lung tumors. We determined the 100 top-ranked marker genes for adenocarcinoma, squamous cell, large cell, and carcinoid using nearest neighbor analysis. Results were validated by immunostaining for 11 selected proteins using a tissue microarray representing 80 tumors. Gene expression data of lung development were accessed from a publicly available dataset generated with the murine Mu11k genome microarray. Self-organized mapping identified two temporally distinct clusters of murine orthologues. Supervised clustering of lung development data showed large-cell carcinoma gene orthologues were in a cluster expressed in pseudoglandular and canalicular stages whereas adenocarcinoma homologues were predominantly in a cluster expressed later in the terminal sac and alveolar stages of murine lung development. Representative large-cell genes (E2F3, MYBL2, HDAC2, CDK4, PCNA) are expressed in the nucleus and are associated with cell cycle and proliferation. In contrast, adenocarcinoma genes are associated with lung-specific transcription pathways (SFTPB, TTF-1), cell adhesion, and signal transduction. In sum, non-small-cell lung tumors histology gene profiles suggest mechanisms relevant to ontogeny and clinical course. Adenocarcinoma genes are associated with differentiation and glandular formation whereas large-cell genes are associated with proliferation and differentiation arrest. The identification of developmentally regulated pathways active in tumorigenesis provides insights into lung carcinogenesis and suggests early steps may differ according to the eventual tumor morphology. PMID:14578194

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit

PubMed Central

Janssen, Bart J; Thodey, Kate; Schaffer, Robert J; Alba, Rob; Balakrishnan, Lena; Bishop, Rebecca; Bowen, Judith H; Crowhurst, Ross N; Gleave, Andrew P; Ledger, Susan; McArtney, Steve; Pichler, Franz B; Snowden, Kimberley C; Ward, Shayna

2008-01-01

Background Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45–55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. Results Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. Conclusion Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development. PMID:18279528

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit.

PubMed

Janssen, Bart J; Thodey, Kate; Schaffer, Robert J; Alba, Rob; Balakrishnan, Lena; Bishop, Rebecca; Bowen, Judith H; Crowhurst, Ross N; Gleave, Andrew P; Ledger, Susan; McArtney, Steve; Pichler, Franz B; Snowden, Kimberley C; Ward, Shayna

2008-02-17

Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

The genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute exposure to the androgen, 17β-trenbolone

USGS Publications Warehouse

Dorts, Jennifer; Richter, Catherine A.; Wright-Osment, Maureen K.; Ellersieck, Mark R.; Carter, Barbara J.; Tillitt, Donald E.

2009-01-01

We investigated the genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute (4 days) exposure to 0.1 or 1.0 ??g/L of 17??-trenbolone (TB), the active metabolite of an anabolic androgenic steroid used as a growth promoter in cattle and a contaminant of concern in aquatic systems. Our objectives were to investigate the gene expression profile induced by TB, define biomarkers of exposure to TB, and increase our understanding of the mechanisms of adverse effects of TB on fish reproduction. In female gonad tissue, microarray analysis using a 22 K oligonucleotide microarray (EcoArray Inc., Gainesville, FL) showed 99 significantly upregulated genes and 741 significantly downregulated genes in response to 1 ??g TB/L. In particular, hydroxysteroid (17??) dehydrogenase 12a (hsd17b12a), zona pellucida glycoprotein 2.2 (zp2.2), and protein inhibitor of activated STAT, 2 (pias2) were all downregulated in gonad. Q-PCR measurements in a larger sample set were consistent with the microarray results in the direction and magnitude of these changes in gene expression. However, several novel potential biomarkers were verified by Q-PCR in the same samples, but could not be validated in independent samples. In liver, Q-PCR measurements showed a significant decrease in vitellogenin 1 (vtg1) mRNA expression. In brain, cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b, previously known as aromatase B) transcript levels were significantly reduced following TB exposure. Our study provides a candidate gene involved in mediating the action of TB, hsd17b12a, and two potential biomarkers sensitive to acute TB exposure, hepatic vtg1 and brain cyp19a1b.

Parasite-specific immune response in adult Drosophila melanogaster: a genomic study

PubMed Central

Roxström-Lindquist, Katarina; Terenius, Olle; Faye, Ingrid

2004-01-01

Insects of the order Diptera are vectors for parasitic diseases such as malaria, sleeping sickness and leishmania. In the search for genes encoding proteins involved in the antiparasitic response, we have used the protozoan parasite Octosporea muscaedomesticae for oral infections of adult Drosophila melanogaster. To identify parasite-specific response molecules, other flies were exposed to virus, bacteria or fungi in parallel. Analysis of gene expression patterns after 24 h of microbial challenge, using Affymetrix oligonucleotide microarrays, revealed a high degree of microbe specificity. Many serine proteases, key intermediates in the induction of insect immune responses, were uniquely expressed following infection of the different organisms. Several lysozyme genes were induced in response to Octosporea infection, while in other treatments they were not induced or downregulated. This suggests that lysozymes are important in antiparasitic defence. PMID:14749722

Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

PubMed

Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

2018-02-01

Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

Visualizing Microbial Biogeochemistry: NanoSIMS and Stable Isotope Probing (Invited)

NASA Astrophysics Data System (ADS)

Pett-Ridge, J.; Weber, P. K.

2009-12-01

Linking phylogenetic information to function in microbial communities is a key challenge for microbial ecology. Isotope-labeling experiments provide a useful means to investigate the ecophysiology of microbial populations and cells in the environment and allow measurement of nutrient transfers between cell types, symbionts and consortia. The combination of Nano-Secondary Ion Mass Spectrometry (NanoSIMS) analysis, in situ labeling and high resolution microscopy allows isotopic analysis to be linked to phylogeny and morphology and holds great promise for fine-scale studies of microbial systems. In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs+, O-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio ‘map’ can then be generated for the analyzed area. NanoSIMS images of 13C, 15N and Mo (a nitrogenase co-factor) localization in diazotrophic cyanobacteria show how cells differentially allocate resources within filaments and allow calculation of nutrient uptake rates on a cell by cell basis. Images of AM fungal hyphae-root and cyanobacteria-rhizobia associations indicate the mobilization and sharing (stealing?) of newly fixed C and N. In a related technique, “El-FISH”, stable isotope labeled biomass is probed with oligonucleotide-elemental labels and then imaged by NanoSIMS. In microbial consortia and cyanobacterial mats, this technique helps link microbial structure and function simultaneously even in systems with unknown and uncultivated microbes. Finally, the combination of re-engineered universal 16S oligonucleotide microarrays with NanoSIMS analyses may allow microbial identity to be linked to functional roles in complex systems such as mats and cellulose degrading hindgut communities. These newly developed methods provide correlated oligonucleotide, functional enzyme and metabolic image data and should help unravel the metabolic processes of complex microbial communities in soils, biofilms and aquatic systems.

Development and Use of Integrated Microarray-Based Genomic Technologies for Assessing Microbial Community Composition and Dynamics

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

Zhou, J.; Wu, L.; Gentry, T.

2006-04-05

To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approximately 2000 unique and group-specific probes with <85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50 C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appearedmore » to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ({approx}1.3{sup o} 10{sup 7} cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r{sup 2} = 0.95-0.99). Application of this microarray to naphthalene-amended enrichments and soil microcosms demonstrated that composition of the microflora varied depending on incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in enrichments, the genes involved in naphthalene degradation from Gram-negative microorganisms such as Ralstonia, Comamonas, and Burkholderia were most abundant in the soil microcosms (as well as those for polyaromatic hydrocarbon and nitrotoluene degradation). Although naphthalene degradation is widely known and studied in Pseudomonas, Pseudomonas genes were not detected in either system. Real-time PCR analysis of 4 representative genes was consistent with microarray-based quantification (r{sup 2} = 0.95). Currently, we are also applying this microarray to the study of several different microbial communities and processes at the NABIR-FRC in Oak Ridge, TN. One project involves the monitoring of the development and dynamics of the microbial community of a fluidized bed reactor (FBR) used for reducing nitrate and the other project monitors microbial community responses to stimulation of uranium reducing populations via ethanol donor additions in situ and in a model system. Additionally, we are developing novel strategies for increasing microarray hybridization sensitivity. Finally, great improvements to our methods of probe design were made by the development of a new computer program, CommOligo. CommOligo designs unique and group-specific oligo probes for whole-genomes, metagenomes, and groups of environmental sequences and uses a new global alignment algorithm to design single or multiple probes for each gene or group. We are now using this program to design a more comprehensive functional gene array for environmental studies. Overall, our results indicate that the 50mer-based microarray technology has potential as a specific and quantitative tool to reveal the composition of microbial communities and their dynamics important to processes within contaminated environments.« less

Gene expression profile of blood cells for the prediction of delayed cerebral ischemia after intracranial aneurysm rupture: a pilot study in humans.

PubMed

Baumann, Antoine; Devaux, Yvan; Audibert, Gérard; Zhang, Lu; Bracard, Serge; Colnat-Coulbois, Sophie; Klein, Olivier; Zannad, Faiez; Charpentier, Claire; Longrois, Dan; Mertes, Paul-Michel

2013-01-01

Delayed cerebral ischemia (DCI) is a potentially devastating complication after intracranial aneurysm rupture and its mechanisms remain poorly elucidated. Early identification of the patients prone to developing DCI after rupture may represent a major breakthrough in its prevention and treatment. The single gene approach of DCI has demonstrated interest in humans. We hypothesized that whole genome expression profile of blood cells may be useful for better comprehension and prediction of aneurysmal DCI. Over a 35-month period, 218 patients with aneurysm rupture were included in this study. DCI was defined as the occurrence of a new delayed neurological deficit occurring within 2 weeks after aneurysm rupture with evidence of ischemia either on perfusion-diffusion MRI, CT angiography or CT perfusion imaging, or with cerebral angiography. DCI patients were matched against controls based on 4 out of 5 criteria (age, sex, Fisher grade, aneurysm location and smoking status). Genome-wide expression analysis of blood cells obtained at admission was performed by microarrays. Transcriptomic analysis was performed using long oligonucleotide microarrays representing 25,000 genes. Quantitative PCR: 1 µg of total RNA extracted was reverse-transcribed, and the resulting cDNA was diluted 10-fold before performing quantitative PCR. Microarray data were first analyzed by 'Significance Analysis of Microarrays' software which includes the Benjamini correction for multiple testing. In a second step, microarray data fold change was compared using a two-tailed, paired t test. Analysis of receiver-operating characteristic (ROC) curves and the area under the ROC curves were used for prediction analysis. Logistic regression models were used to investigate the additive value of multiple biomarkers. A total of 16 patients demonstrated DCI. Significance Analysis of Microarrays software failed to retrieve significant genes, most probably because of the heterogeneity of the patients included in the microarray experiments and the small size of the DCI population sample. Standard two-tailed paired t test and C-statistic revealed significant associations between gene expression and the occurrence of DCI: in particular, the expression of neuroregulin 1 was 1.6-fold upregulated in patients with DCI (p = 0.01) and predicted DCI with an area under the ROC curve of 0.96. Logistic regression analyses revealed a significant association between neuroregulin 1 and DCI (odds ratio 1.46, 95% confidence interval 1.02-2.09, p = 0.02). This pilot study suggests that blood cells may be a reservoir of prognostic biomarkers of DCI in patients with intracranial aneurysm rupture. Despite an evident lack of power, this study elicited neuroregulin 1, a vasoreactivity-, inflammation- and angiogenesis-related gene, as a possible candidate predictor of DCI. Larger cohort studies are needed but genome-wide microarray-based studies are promising research tools for the understanding of DCI after intracranial aneurysm rupture. © 2013 S. Karger AG, Basel.

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

PubMed Central

Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

2015-01-01

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ. PMID:26240388

Fluorogenic PNA probes

PubMed Central

2018-01-01

Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years. PMID:29507634

BABAR: an R package to simplify the normalisation of common reference design microarray-based transcriptomic datasets

PubMed Central

2010-01-01

Background The development of DNA microarrays has facilitated the generation of hundreds of thousands of transcriptomic datasets. The use of a common reference microarray design allows existing transcriptomic data to be readily compared and re-analysed in the light of new data, and the combination of this design with large datasets is ideal for 'systems'-level analyses. One issue is that these datasets are typically collected over many years and may be heterogeneous in nature, containing different microarray file formats and gene array layouts, dye-swaps, and showing varying scales of log2- ratios of expression between microarrays. Excellent software exists for the normalisation and analysis of microarray data but many data have yet to be analysed as existing methods struggle with heterogeneous datasets; options include normalising microarrays on an individual or experimental group basis. Our solution was to develop the Batch Anti-Banana Algorithm in R (BABAR) algorithm and software package which uses cyclic loess to normalise across the complete dataset. We have already used BABAR to analyse the function of Salmonella genes involved in the process of infection of mammalian cells. Results The only input required by BABAR is unprocessed GenePix or BlueFuse microarray data files. BABAR provides a combination of 'within' and 'between' microarray normalisation steps and diagnostic boxplots. When applied to a real heterogeneous dataset, BABAR normalised the dataset to produce a comparable scaling between the microarrays, with the microarray data in excellent agreement with RT-PCR analysis. When applied to a real non-heterogeneous dataset and a simulated dataset, BABAR's performance in identifying differentially expressed genes showed some benefits over standard techniques. Conclusions BABAR is an easy-to-use software tool, simplifying the simultaneous normalisation of heterogeneous two-colour common reference design cDNA microarray-based transcriptomic datasets. We show BABAR transforms real and simulated datasets to allow for the correct interpretation of these data, and is the ideal tool to facilitate the identification of differentially expressed genes or network inference analysis from transcriptomic datasets. PMID:20128918

Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

PubMed Central

Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

2014-01-01

The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

Perspectives of gene expression profiling for diagnosis and therapy in haematological malignancies.

PubMed

Bacher, Ulrike; Kohlmann, Alexander; Haferlach, Torsten

2009-05-01

Considering the heterogeneity of leukaemias and the widening spectrum of therapeutic strategies, novel diagnostic methods are urgently needed for haematological malignancies. For a decade, gene expression profiling (GEP) has been applied in leukaemia research. Thus, various studies demonstrated worldwide that the majority of genetically defined leukaemia subtypes are accurately predictable by GEP, for example, with respect to reciprocal rearrangements in acute myeloid leukaemia (AML). Moreover, novel prognostically relevant gene classifiers were developed as, for example, in normal karyotype AML. Considering the lymphatic malignancies, GEP studies defined novel clinically relevant subtypes in diffuse large B cell lymphoma (DLBCL), and improved the discrimination of Burkitt lymphoma and DLBCL cases, overcoming considerable overlaps of these entities that exist from morphological and genetic perspectives. Treatment-specific sensitivity assays are being developed for targeted drugs such as farnesyl transferase inhibitors in AML or imatinib in BCR-ABL1 positive acute lymphoblastic leukaemia (ALL). Irrespectively of these proceedings, an introduction of the microarray technology in haematological practice requires diagnostic algorithms and strategies for interaction with currently established diagnostic techniques. Large multicentre studies such as the MILE Study (Microarray Innovations in LEukemia) aim at translating this methodology into clinical routine workflows and to catalyze this process.

Quality control of inkjet technology for DNA microarray fabrication.

PubMed

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

2008-12-01

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

Transcriptional response to 131I exposure of rat thyroid gland.

PubMed

Rudqvist, Nils; Spetz, Johan; Schüler, Emil; Parris, Toshima Z; Langen, Britta; Helou, Khalil; Forssell-Aronsson, Eva

2017-01-01

Humans are exposed to 131I in medical diagnostics and treatment but also from nuclear accidents, and better knowledge of the molecular response in thyroid is needed. The aim of the study was to examine the transcriptional response in thyroid tissue 24 h after 131I administration in rats. The exposure levels were chosen to simulate both the clinical situation and the case of nuclear fallout. Thirty-six male rats were i.v. injected with 0-4700 kBq 131I, and killed at 24 h after injection (Dthyroid = 0.0058-3.0 Gy). Total RNA was extracted from individual thyroid tissue samples and mRNA levels were determined using oligonucleotide microarray technique. Differentially expressed transcripts were determined using Nexus Expression 3.0. Hierarchical clustering was performed in the R statistical computing environment. Pathway analysis was performed using the Ingenuity Pathway Analysis tool and the Gene Ontology database. T4 and TSH plasma concentrations were measured using ELISA. Totally, 429 differentially regulated transcripts were identified. Downregulation of thyroid hormone biosynthesis associated genes (e.g. thyroglobulin, thyroid peroxidase, the sodium-iodine symporter) was identified in some groups, and an impact on thyroid function was supported by the pathway analysis. Recurring downregulation of Dbp and Slc47a2 was found. Dbp exhibited a pattern with monotonous reduction of downregulation with absorbed dose at 0.0058-0.22 Gy. T4 plasma levels were increased and decreased in rats whose thyroids were exposed to 0.057 and 0.22 Gy, respectively. Different amounts of injected 131I gave distinct transcriptional responses in the rat thyroid. Transcriptional response related to thyroid function and changes in T4 plasma levels were found already at very low absorbed doses to thyroid.

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

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

PubMed Central

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

2004-01-01

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

Expanding probe repertoire and improving reproducibility in human genomic hybridization

PubMed Central

Dorman, Stephanie N.; Shirley, Ben C.; Knoll, Joan H. M.; Rogan, Peter K.

2013-01-01

Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance. A divide and conquer algorithm was implemented to design sc probes. With this approach, sc probes can include divergent repetitive elements, which hybridize to unique genomic targets under higher stringency experimental conditions. Genome-wide custom probe sets were created for fluorescent in situ hybridization (FISH) and microarray genomic hybridization. The scFISH probes were developed for detection of copy number changes within small tumour suppressor genes and oncogenes. The microarrays demonstrated increased reproducibility by eliminating cross-hybridization to repetitive sequences adjacent to probe targets. The genome-wide microarrays exhibited lower median coefficients of variation (17.8%) for two HapMap family trios. The coefficients of variations of commercial probes within 300 nt of a repetitive element were 48.3% higher than the nearest custom probe. Furthermore, the custom microarray called a chromosome 15q11.2q13 deletion more consistently. This method for sc probe design increases probe coverage for FISH and lowers variability in genomic microarrays. PMID:23376933

Genes Responsive to Low-Intensity Pulsed Ultrasound in MC3T3-E1 Preosteoblast Cells

PubMed Central

Tabuchi, Yoshiaki; Sugahara, Yuuki; Ikegame, Mika; Suzuki, Nobuo; Kitamura, Kei-ichiro; Kondo, Takashi

2013-01-01

Although low-intensity pulsed ultrasound (LIPUS) has been shown to enhance bone fracture healing, the underlying mechanism of LIPUS remains to be fully elucidated. Here, to better understand the molecular mechanism underlying cellular responses to LIPUS, we investigated gene expression profiles in mouse MC3T3-E1 preosteoblast cells exposed to LIPUS using high-density oligonucleotide microarrays and computational gene expression analysis tools. Although treatment of the cells with a single 20-min LIPUS (1.5 MHz, 30 mW/cm2) did not affect the cell growth or alkaline phosphatase activity, the treatment significantly increased the mRNA level of Bglap. Microarray analysis demonstrated that 38 genes were upregulated and 37 genes were downregulated by 1.5-fold or more in the cells at 24-h post-treatment. Ingenuity pathway analysis demonstrated that the gene network U (up) contained many upregulated genes that were mainly associated with bone morphology in the category of biological functions of skeletal and muscular system development and function. Moreover, the biological function of the gene network D (down), which contained downregulated genes, was associated with gene expression, the cell cycle and connective tissue development and function. These results should help to further clarify the molecular basis of the mechanisms of the LIPUS response in osteoblast cells. PMID:24252911

Identification of Cell Cycle-regulated Genes in Fission YeastD⃞

PubMed Central

Peng, Xu; Karuturi, R. Krishna Murthy; Miller, Lance D.; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T.; Balasubramanian, Mohan K.; Liu, Jianhua

2005-01-01

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC. PMID:15616197

Methoxychlor affects multiple hormone signaling pathways in the largemouth bass (Micropterus salmoides) liver

PubMed Central

Martyniuk, Christopher J.; Spade, Daniel J.; Blum, Jason L.; Kroll, Kevin J.; Denslow, Nancy D.

2011-01-01

Methoxychlor (MXC) is an organochlorine pesticide that has been shown to have estrogenic activity by activating estrogen receptors and inducing vitellogenin production in male fish. Previous studies report that exposure to MXC induces changes in mRNA abundance of reproductive genes in the liver and testes of largemouth bass (Micropterus salmoides). The objective of the present study was to better characterize the mode of action of MXC by measuring the global transcriptomic response in the male largemouth liver using an oligonucleotide microarray. Microarray analysis identified highly significant changes in the expression of 37 transcripts (p<0.001) (20 induced and 17 decreased) in the liver after MXC injection and a total of 900 expression changes (p<0.05) in transcripts with high homology to known genes. Largemouth bass estrogen receptor alpha (esr1) and androgen receptor (ar) were among the transcripts that were increased in the liver after MXC treatment. Functional enrichment analysis identified the molecular functions of steroid binding and androgen receptor activity as well as steroid hormone receptor activity as being significantly over-represented gene ontology terms. Pathway analysis identified c-fos signaling as being putatively affected through both estrogen and androgen signaling. This study provides evidence that MXC elicits transcriptional effects through the estrogen receptor as well as androgen receptor-mediated pathways in the liver. PMID:21276474

Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators.

PubMed

Klix, V; Nowrousian, M; Ringelberg, C; Loros, J J; Dunlap, J C; Pöggeler, S

2010-06-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators▿†

PubMed Central

Klix, V.; Nowrousian, M.; Ringelberg, C.; Loros, J. J.; Dunlap, J. C.; Pöggeler, S.

2010-01-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes. PMID:20435701

Response of Desulfovibrio vulgaris to Alkaline Stress▿ †

PubMed Central

Stolyar, Sergey; He, Qiang; Joachimiak, Marcin P.; He, Zhili; Yang, Zamin Koo; Borglin, Sharon E.; Joyner, Dominique C.; Huang, Katherine; Alm, Eric; Hazen, Terry C.; Zhou, Jizhong; Wall, Judy D.; Arkin, Adam P.; Stahl, David A.

2007-01-01

The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580). PMID:17921288

Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays

PubMed Central

Sugnet, Charles W; Srinivasan, Karpagam; Clark, Tyson A; O'Brien, Georgeann; Cline, Melissa S; Wang, Hui; Williams, Alan; Kulp, David; Blume, John E; Haussler, David; Ares, Manuel

2006-01-01

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families. PMID:16424921

Transcriptome remodeling associated with chronological aging in the dinoflagellate, Karenia brevis.

PubMed

Johnson, Jillian G; Morey, Jeanine S; Neely, Marion G; Ryan, James C; Van Dolah, Frances M

2012-03-01

The toxic dinoflagellate, Karenia brevis, forms dense blooms in the Gulf of Mexico that persist for many months in coastal waters, where they can cause extensive marine animal mortalities and human health impacts. The mechanisms that enable cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. To gain an understanding of processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out to identify changes in the global transcriptome that accompany the entry and maintenance of stationary phase up to the onset of cell death. The transcriptome of K. brevis was assayed using a custom 10,263 feature oligonucleotide microarray from mid-logarithmic growth to the onset of culture demise. A total of 2958 (29%) features were differentially expressed, with the mid-stationary phase timepoint demonstrating peak changes in expression. Gene ontology enrichment analyses identified a significant shift in transcripts involved in energy acquisition, ribosome biogenesis, gene expression, stress adaptation, calcium signaling, and putative brevetoxin biosynthesis. The extensive remodeling of the transcriptome observed in the transition into a quiescent non-dividing phase appears to be indicative of a global shift in the metabolic and signaling requirements and provides the basis from which to understand the process of chronological aging in a dinoflagellate. Published by Elsevier B.V.

Application of an oligonucleotide microarray-based nano-amplification technique for the detection of fungal pathogens.

PubMed

Lu, Weiping; Gu, Dayong; Chen, Xingyun; Xiong, Renping; Liu, Ping; Yang, Nan; Zhou, Yuanguo

2010-10-01

The traditional techniques for diagnosis of invasive fungal infections in the clinical microbiology laboratory need improvement. These techniques are prone to delay results due to their time-consuming process, or result in misidentification of the fungus due to low sensitivity or low specificity. The aim of this study was to develop a method for the rapid detection and identification of fungal pathogens. The internal transcribed spacer two fragments of fungal ribosomal DNA were amplified using a polymerase chain reaction for all samples. Next, the products were hybridized with the probes immobilized on the surface of a microarray. These species-specific probes were designed to detect nine different clinical pathogenic fungi including Candida albicans, Candida tropocalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida guilliermondii, Candida keyfr, and Cryptococcus neoformans. The hybridizing signals were enhanced with gold nanoparticles and silver deposition, and detected using a flatbed scanner or visually. Fifty-nine strains of fungal pathogens, including standard and clinically isolated strains, were correctly identified by this method. The sensitivity of the assay for Candida albicans was 10 cells/mL. Ten cultures from clinical specimens and 12 clinical samples spiked with fungi were also identified correctly. This technique offers a reliable alternative to conventional methods for the detection and identification of fungal pathogens. It has higher efficiency, specificity and sensitivity compared with other methods commonly used in the clinical laboratory.

Bioorthogonal in Situ Hydrogels Based on Polyether Polyols for New Biosensor Materials with High Sensitivity.

PubMed

Herrmann, Anna; Kaufmann, Lena; Dey, Pradip; Haag, Rainer; Schedler, Uwe

2018-04-04

Both noncovalent and covalent encapsulations of active biomolecules, for example, proteins and oligonucleotides, for a new biosensor matrix in an in situ bioorthogonal hydrogel formation via a strain-promoted azide-alkyne cycloaddition reaction were investigated. Unspecific interaction between the gel and the biomolecules as well as protein denaturation was prevented by the bioorthogonal gel components, which ensure a uniform aqueous environment in the hydrogel network. No leaching of the active biomolecules was observed. Additionally, a much higher and also adjustable loading of biomolecules in the hydrogel matrix was achieved compared to conventional biosensor surfaces, where the sensor molecules are immobilized on monolayers (2D surfaces) or brushlike structures (3D surfaces). Spotting experiments of the hydrogel confirm the possibility to use this new surface for microarray-based multiplex applications which require very high signal-to-noise ratios.

Multiplex amplification of large sets of human exons.

PubMed

Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

2007-11-01

A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

Microfabricated Fountain Pens for High-Density DNA Arrays

PubMed Central

Reese, Matthew O.; van Dam, R. Michae; Scherer, Axel; Quake, Stephen R.

2003-01-01

We used photolithographic microfabrication techniques to create very small stainless steel fountain pens that were installed in place of conventional pens on a microarray spotter. Because of the small feature size produced by the microfabricated pens, we were able to print arrays with up to 25,000 spots/cm2, significantly higher than can be achieved by other deposition methods. This feature density is sufficiently large that a standard microscope slide can contain multiple replicates of every gene in a complex organism such as a mouse or human. We tested carryover during array printing with dye solution, labeled DNA, and hybridized DNA, and we found it to be indistinguishable from background. Hybridization also showed good sequence specificity to printed oligonucleotides. In addition to improved slide capacity, the microfabrication process offers the possibility of low-cost mass-produced pens and the flexibility to include novel pen features that cannot be machined with conventional techniques. PMID:12975313

Aberrant expression of the PHF14 gene in biliary tract cancer cells

PubMed Central

AKAZAWA, TAKAKO; YASUI, KOHICHIROH; GEN, YASUYUKI; YAMADA, NOBUHISA; TOMIE, AKIRA; DOHI, OSAMU; MITSUYOSHI, HIRONORI; YAGI, NOBUAKI; ITOH, YOSHITO; NAITO, YUJI; YOSHIKAWA, TOSHIKAZU

2013-01-01

DNA copy number aberrations in human biliary tract cancer (BTC) cell lines were investigated using a high-density oligonucleotide microarray. A novel homozygous deletion was detected at chromosomal region 7p21.3 in the OZ cell line. Further validation experiments using genomic PCR revealed a homozygous deletion of a single gene, plant homeodomain (PHD) finger protein 14 (PHF14). No PHF14 mRNA or protein expression was detected, thus demonstrating the absence of PHF14 expression in the OZ cell line. Although the PHD finger protein is considered to be involved in chromatin-mediated transcriptional regulation, little is known about the function of PHF14 in cancer. The present study observed that the knock down of PHF14 using small interfering RNA (siRNA) enhanced the growth of the BTC cells. These observations suggest that aberrant PHF14 expression may have a role in the tumorigenesis of BTC. PMID:23833654

ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds

PubMed Central

Osaka, Nao; Takahashi, Takumi; Murakami, Shiori; Matsuzawa, Atsushi; Noguchi, Takuya; Fujiwara, Takeshi; Aburatani, Hiroyuki; Moriyama, Keiji; Takeda, Kohsuke; Ichijo, Hidenori

2007-01-01

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein 3-kinase family that activates both c-Jun NH2-terminal kinase and p38 pathways in response to inflammatory cytokines and physicochemical stress. We report that ASK1 deficiency in mice results in dramatic retardation of wounding-induced hair regrowth in skin. Oligonucleotide microarray analysis revealed that expression of several chemotactic and activating factors for macrophages, as well as several macrophage-specific marker genes, was reduced in the skin wound area of ASK1-deficient mice. Intracutaneous transplantation of cytokine-activated bone marrow-derived macrophages strongly induced hair growth in both wild-type and ASK1-deficient mice. These findings indicate that ASK1 is required for wounding-induced infiltration and activation of macrophages, which play central roles in inflammation-dependent hair regrowth in skin. PMID:17389227

Gene expression profiling in the hippocampus of learned helpless and nonhelpless rats.

PubMed

Kohen, R; Kirov, S; Navaja, G P; Happe, H Kevin; Hamblin, M W; Snoddy, J R; Neumaier, J F; Petty, F

2005-01-01

In the learned helplessness (LH) animal model of depression, failure to attempt escape from avoidable environmental stress, LH, indicates behavioral despair, whereas nonhelpless (NH) behavior reflects behavioral resilience to the effects of environmental stress. Comparing hippocampal gene expression with large-scale oligonucleotide microarrays, we found that stress-resilient (NH) rats, although behaviorally indistinguishable from controls, showed a distinct gene expression profile compared to LH, sham stressed, and naïve control animals. Genes that were confirmed as differentially expressed in the NH group by quantitative PCR strongly correlated in their levels of expression across all four animal groups. Differential expression could not be confirmed at the protein level. We identified several shared degenerate sequence motifs in the 3' untranslated region (3'UTR) of differentially expressed genes that could be a factor in this tight correlation of expression levels among differentially expressed genes.

Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus.

PubMed

Bogestam, Katja; Vondracek, Martin; Karlsson, Mattias; Fang, Hong; Giske, Christian G

2018-01-01

Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE.

PubMed

Rao, Archana N; Grainger, David W

2014-04-01

Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA's persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools.

BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

PubMed Central

Rao, Archana N.; Grainger, David W.

2014-01-01

Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA’s persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools. PMID:24765522

New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

PubMed

Gbaj, A; Bichenkova, Ev; Walsh, L; Savage, He; Sardarian, Ar; Etchells, Ll; Gulati, A; Hawisa, S; Douglas, Kt

2009-12-01

The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5'-bispyrene and 3'-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5'-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices.

PubMed

Knob, Radim; Hanson, Robert L; Tateoka, Olivia B; Wood, Ryan L; Guerrero-Arguero, Israel; Robison, Richard A; Pitt, William G; Woolley, Adam T

2018-05-21

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection. Copyright © 2018 Elsevier B.V. All rights reserved.

76 FR 4921 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-27

... chemical imaging (IRCI) to detect nanostructure-based DNA microarrays, which can be utilized in the life science research arena to examine gene expression and single nucleotide polymorphisms (SNPs), as well as... can be applied to the areas of environmental sciences, agriculture research, bio-defense, diagnostics...

Visualization of genome signatures of eukaryote genomes by batch-learning self-organizing map with a special emphasis on Drosophila genomes.

PubMed

Abe, Takashi; Hamano, Yuta; Ikemura, Toshimichi

2014-01-01

A strategy of evolutionary studies that can compare vast numbers of genome sequences is becoming increasingly important with the remarkable progress of high-throughput DNA sequencing methods. We previously established a sequence alignment-free clustering method "BLSOM" for di-, tri-, and tetranucleotide compositions in genome sequences, which can characterize sequence characteristics (genome signatures) of a wide range of species. In the present study, we generated BLSOMs for tetra- and pentanucleotide compositions in approximately one million sequence fragments derived from 101 eukaryotes, for which almost complete genome sequences were available. BLSOM recognized phylotype-specific characteristics (e.g., key combinations of oligonucleotide frequencies) in the genome sequences, permitting phylotype-specific clustering of the sequences without any information regarding the species. In our detailed examination of 12 Drosophila species, the correlation between their phylogenetic classification and the classification on the BLSOMs was observed to visualize oligonucleotides diagnostic for species-specific clustering.

Carbon Nanotube Nanoelectrode Array for Ultrasensitive DNA Detection

NASA Technical Reports Server (NTRS)

Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

2003-01-01

A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6. The open-end of MWNTs present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. Oligonucleotide probes are selectively functionalized at the open ends cf the nanotube array and specifically hybridized with oligonucleotide targets. The guanine groups are employed as the signal moieties in the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. The hybridization of subattomoles of PCR amplified DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the Ru(bpy)32' amplification mechanism. This system provides a general platform of molecular diagnostics for applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparations.

Development and characterization of a microheater array device for real-time DNA mutation detection

NASA Astrophysics Data System (ADS)

Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

2008-04-01

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Development and characterization of a microheater array device for real-time DNA mutation detection

NASA Astrophysics Data System (ADS)

Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

2008-02-01

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

PubMed

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Polar bear encephalitis: establishment of a comprehensive next-generation pathogen analysis pipeline for captive and free-living wildlife.

PubMed

Szentiks, C A; Tsangaras, K; Abendroth, B; Scheuch, M; Stenglein, M D; Wohlsein, P; Heeger, F; Höveler, R; Chen, W; Sun, W; Damiani, A; Nikolin, V; Gruber, A D; Grobbel, M; Kalthoff, D; Höper, D; Czirják, G Á; Derisi, J; Mazzoni, C J; Schüle, A; Aue, A; East, M L; Hofer, H; Beer, M; Osterrieder, N; Greenwood, A D

2014-05-01

This report describes three possibly related incidences of encephalitis, two of them lethal, in captive polar bears (Ursus maritimus). Standard diagnostic methods failed to identify pathogens in any of these cases. A comprehensive, three-stage diagnostic 'pipeline' employing both standard serological methods and new DNA microarray and next generation sequencing-based diagnostics was developed, in part as a consequence of this initial failure. This pipeline approach illustrates the strengths, weaknesses and limitations of these tools in determining pathogen caused deaths in non-model organisms such as wildlife species and why the use of a limited number of diagnostic tools may fail to uncover important wildlife pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

PubMed

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

2008-01-01

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

Microarrays: Molecular allergology and nanotechnology for personalised medicine (II).

PubMed

Lucas, J M

2010-01-01

Progress in nanotechnology and DNA recombination techniques have produced tools for the diagnosis and investigation of allergy at molecular level. The most advanced examples of such progress are the microarray techniques, which have been expanded not only in research in the field of proteomics but also in application to the clinical setting. Microarrays of allergic components offer results relating to hundreds of allergenic components in a single test, and using a small amount of serum which can be obtained from capillary blood. The availability of new molecules will allow the development of panels including new allergenic components and sources, which will require evaluation for clinical use. Their application opens the door to component-based diagnosis, to the holistic perception of sensitisation as represented by molecular allergy, and to patient-centred medical practice by allowing great diagnostic accuracy and the definition of individualised immunotherapy for each patient. The present article reviews the application of allergenic component microarrays to allergology for diagnosis, management in the form of specific immunotherapy, and epidemiological studies. A review is also made of the use of protein and gene microarray techniques in basic research and in allergological diseases. Lastly, an evaluation is made of the challenges we face in introducing such techniques to clinical practice, and of the future perspectives of this new technology. Copyright 2010 SEICAP. Published by Elsevier Espana. All rights reserved.

A hybrid approach to device integration on a genetic analysis platform

NASA Astrophysics Data System (ADS)

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

2012-10-01

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

Multiple biomarkers in molecular oncology. II. Molecular diagnostics applications in breast cancer management.

PubMed

Malinowski, Douglas P

2007-05-01

In recent years, the application of genomic and proteomic technologies to the problem of breast cancer prognosis and the prediction of therapy response have begun to yield encouraging results. Independent studies employing transcriptional profiling of primary breast cancer specimens using DNA microarrays have identified gene expression profiles that correlate with clinical outcome in primary breast biopsy specimens. Recent advances in microarray technology have demonstrated reproducibility, making clinical applications more achievable. In this regard, one such DNA microarray device based upon a 70-gene expression signature was recently cleared by the US FDA for application to breast cancer prognosis. These DNA microarrays often employ at least 70 gene targets for transcriptional profiling and prognostic assessment in breast cancer. The use of PCR-based methods utilizing a small subset of genes has recently demonstrated the ability to predict the clinical outcome in early-stage breast cancer. Furthermore, protein-based immunohistochemistry methods have progressed from using gene clusters and gene expression profiling to smaller subsets of expressed proteins to predict prognosis in early-stage breast cancer. Beyond prognostic applications, DNA microarray-based transcriptional profiling has demonstrated the ability to predict response to chemotherapy in early-stage breast cancer patients. In this review, recent advances in the use of multiple markers for prognosis of disease recurrence in early-stage breast cancer and the prediction of therapy response will be discussed.

Autonomous system for Web-based microarray image analysis.

PubMed

Bozinov, Daniel

2003-12-01

Software-based feature extraction from DNA microarray images still requires human intervention on various levels. Manual adjustment of grid and metagrid parameters, precise alignment of superimposed grid templates and gene spots, or simply identification of large-scale artifacts have to be performed beforehand to reliably analyze DNA signals and correctly quantify their expression values. Ideally, a Web-based system with input solely confined to a single microarray image and a data table as output containing measurements for all gene spots would directly transform raw image data into abstracted gene expression tables. Sophisticated algorithms with advanced procedures for iterative correction function can overcome imminent challenges in image processing. Herein is introduced an integrated software system with a Java-based interface on the client side that allows for decentralized access and furthermore enables the scientist to instantly employ the most updated software version at any given time. This software tool is extended from PixClust as used in Extractiff incorporated with Java Web Start deployment technology. Ultimately, this setup is destined for high-throughput pipelines in genome-wide medical diagnostics labs or microarray core facilities aimed at providing fully automated service to its users.

Evaluation of a low density DNA microarray for small B-cell non-Hodgkin lymphoma differential diagnosis.

PubMed

Gillet, Jean-Pierre; Molina, Thierry Jo; Jamart, Jacques; Gaulard, Philippe; Leroy, Karen; Briere, Josette; Theate, Ivan; Thieblemont, Catherine; Bosly, Andre; Herin, Michel; Hamels, Jacques; Remacle, Jose

2009-03-01

Lymphomas are classified according to the World Health Organisation (WHO) classification which defines subtypes on the basis of clinical, morphological, immunophenotypic, molecular and cytogenetic criteria. Differential diagnosis of the subtypes is sometimes difficult, especially for small B-cell lymphoma (SBCL). Standardisation of molecular genetic assays using multiple gene expression analysis by microarrays could be a useful complement to the current diagnosis. The aim of the present study was to develop a low density DNA microarray for the analysis of 107 genes associated with B-cell non-Hodgkin lymphoma and to evaluate its performance in the diagnosis of SBCL. A predictive tool based on Fisher discriminant analysis using a training set of 40 patients including four different subtypes (follicular lymphoma n = 15, mantle cell lymphoma n = 7, B-cell chronic lymphocytic leukemia n = 6 and splenic marginal zone lymphoma n = 12) was designed. A short additional preliminary analysis to gauge the accuracy of this signature was then performed on an external set of nine patients. Using this model, eight of nine of those samples were classified successfully. This pilot study demonstrates that such a microarray tool may be a promising diagnostic approach for small B-cell non-Hodgkin lymphoma.

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

PubMed Central

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

2012-01-01

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

Evaluation of gene expression classification studies: factors associated with classification performance.

PubMed

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

2014-01-01

Classification methods used in microarray studies for gene expression are diverse in the way they deal with the underlying complexity of the data, as well as in the technique used to build the classification model. The MAQC II study on cancer classification problems has found that performance was affected by factors such as the classification algorithm, cross validation method, number of genes, and gene selection method. In this paper, we study the hypothesis that the disease under study significantly determines which method is optimal, and that additionally sample size, class imbalance, type of medical question (diagnostic, prognostic or treatment response), and microarray platform are potentially influential. A systematic literature review was used to extract the information from 48 published articles on non-cancer microarray classification studies. The impact of the various factors on the reported classification accuracy was analyzed through random-intercept logistic regression. The type of medical question and method of cross validation dominated the explained variation in accuracy among studies, followed by disease category and microarray platform. In total, 42% of the between study variation was explained by all the study specific and problem specific factors that we studied together.

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays

PubMed Central

Fixe, F.; Dufva, M.; Telleman, P.; Christensen, C. B. V.

2004-01-01

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray™ slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques. PMID:14718554

Recommendations for the use of microarrays in prenatal diagnosis.

PubMed

Suela, Javier; López-Expósito, Isabel; Querejeta, María Eugenia; Martorell, Rosa; Cuatrecasas, Esther; Armengol, Lluis; Antolín, Eugenia; Domínguez Garrido, Elena; Trujillo-Tiebas, María José; Rosell, Jordi; García Planells, Javier; Cigudosa, Juan Cruz

2017-04-07

Microarray technology, recently implemented in international prenatal diagnosis systems, has become one of the main techniques in this field in terms of detection rate and objectivity of the results. This guideline attempts to provide background information on this technology, including technical and diagnostic aspects to be considered. Specifically, this guideline defines: the different prenatal sample types to be used, as well as their characteristics (chorionic villi samples, amniotic fluid, fetal cord blood or miscarriage tissue material); variant reporting policies (including variants of uncertain significance) to be considered in informed consents and prenatal microarray reports; microarray limitations inherent to the technique and which must be taken into account when recommending microarray testing for diagnosis; a detailed clinical algorithm recommending the use of microarray testing and its introduction into routine clinical practice within the context of other genetic tests, including pregnancies in families with a genetic history or specific syndrome suspicion, first trimester increased nuchal translucency or second trimester heart malformation and ultrasound findings not related to a known or specific syndrome. This guideline has been coordinated by the Spanish Association for Prenatal Diagnosis (AEDP, «Asociación Española de Diagnóstico Prenatal»), the Spanish Human Genetics Association (AEGH, «Asociación Española de Genética Humana») and the Spanish Society of Clinical Genetics and Dysmorphology (SEGCyD, «Sociedad Española de Genética Clínica y Dismorfología»). Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

PubMed

Marlowe, Jennifer L; Akopian, Violetta; Karmali, Priya; Kornbrust, Douglas; Lockridge, Jennifer; Semple, Sean

2017-08-01

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

Chemically functionalized gold nanoparticles: Synthesis, characterization, and applications

NASA Astrophysics Data System (ADS)

Daniel, Weston Lewis

This thesis focuses on the development and application of gold nanoparticle based detection systems and biomimetic structures. Each class of modified nanoparticle has properties that are defined by its chemical moieties that interface with solution and the gold nanoparticle core. In Chapter 2, a comparison of the biomolecular composition and binding properties of various preparations of antibody oligonucleotide gold nanoparticle conjugates is presented. These constructs differed significantly in terms of their structure and binding properties. Chapter 3 reports the use of electroless gold deposition as a light scattering signal enhancer in a multiplexed, microarray-based scanometric immunoassay using the gold nanoparticle probes evaluated in Chapter 2. The use of gold development results in greater signal enhancement than the typical silver development, and multiple rounds of metal development were found to increase the resulting signal compared to one development. Chapter 4 describes an amplified scanometric detection method for human telomerase activity. Gold nanoparticles functionalized with specific oligonucleotide sequences can efficiently capture telomerase enzymes and subsequently be elongated. Both the elongated and unmodified oligonucleotide sequences are simultaneously measured. At low telomerase concentrations, elongated strands cannot be detected, but the unmodified sequences, which come from the same probe particles, can be detected because their concentration is higher, providing a novel form of amplification. Chapter 5 reports the development of a novel colorimetric nitrite and nitrate ion assay based upon gold nanoparticle probes functionalized with Griess reaction reagents. This assay takes advantage of the distance-dependent plasmonic properties of the gold nanoparticles and the ability of nitrite ion to facilitate the cross coupling of novel nanoparticle probes. The assay works on the concept of a kinetic end point and can be triggered at the EPA limit for this ion in drinking water. Finally, Chapter 6 describes the synthesis of high density lipoprotein biomimetic nanoparticles capable of binding cholesterol. These structures use a gold nanoparticle core to template the assembly of a mixed phospholipid layer and the adsorption of apolipoprotein A-I. These synthesized structures have the general size and surface composition of natural HDL and bind free cholesterol with a Kd of 4 nM.

The delivery of therapeutic oligonucleotides

PubMed Central

Juliano, Rudolph L.

2016-01-01

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. PMID:27084936

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

PubMed

Ershova, Anna S; Gra, Olga A; Lyaschuk, Alexander M; Grunina, Tatyana M; Tkachuk, Artem P; Bartov, Mikhail S; Savina, Darya M; Sergienko, Olga V; Galushkina, Zoya M; Gudov, Vladimir P; Kozlovskaya, Liubov I; Kholodilov, Ivan S; Gmyl, Larissa V; Karganova, Galina G; Lunin, Vladimir G; Karyagina, Anna S; Gintsburg, Alexander L

2016-10-07

E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.

GeoChip 3.0: A High Throughput Tool for Analyzing Microbial Community, Composition, Structure, and Functional Activity

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

He, Zhili; Deng, Ye; Nostrand, Joy Van

2010-05-17

Microarray-based genomic technology has been widely used for microbial community analysis, and it is expected that microarray-based genomic technologies will revolutionize the analysis of microbial community structure, function and dynamics. A new generation of functional gene arrays (GeoChip 3.0) has been developed, with 27,812 probes covering 56,990 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance, and organic contaminant degradation. Those probes were derived from 2,744, 140, and 262 species for bacteria, archaea, and fungi, respectively. GeoChip 3.0 has several other distinct features, such as a common oligomore » reference standard (CORS) for data normalization and comparison, a software package for data management and future updating, and the gyrB gene for phylogenetic analysis. Our computational evaluation of probe specificity indicated that all designed probes had a high specificity to their corresponding targets. Also, experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036percent-0.025percent false positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which demonstrated that the structure, composition, and potential activity of soil microbial communities significantly changed with the plant species diversity. All results indicate that GeoChip 3.0 is a high throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning. To our knowledge, GeoChip 3.0 is the most comprehensive microarrays currently available for studying microbial communities associated with geobiochemical cycling, global climate change, bioenergy, agricuture, land use, ecosystem management, environmental cleanup and restoration, bioreactor systems, and human health.« less

Revisiting adverse effects of cross-hybridization in Affymetrix gene expression data: do they matter for correlation analysis?

PubMed

Klebanov, Lev; Chen, Linlin; Yakovlev, Andrei

2007-11-07

This work was undertaken in response to a recently published paper by Okoniewski and Miller (BMC Bioinformatics 2006, 7: Article 276). The authors of that paper came to the conclusion that the process of multiple targeting in short oligonucleotide microarrays induces spurious correlations and this effect may deteriorate the inference on correlation coefficients. The design of their study and supporting simulations cast serious doubt upon the validity of this conclusion. The work by Okoniewski and Miller drove us to revisit the issue by means of experimentation with biological data and probabilistic modeling of cross-hybridization effects. We have identified two serious flaws in the study by Okoniewski and Miller: (1) The data used in their paper are not amenable to correlation analysis; (2) The proposed simulation model is inadequate for studying the effects of cross-hybridization. Using two other data sets, we have shown that removing multiply targeted probe sets does not lead to a shift in the histogram of sample correlation coefficients towards smaller values. A more realistic approach to mathematical modeling of cross-hybridization demonstrates that this process is by far more complex than the simplistic model considered by the authors. A diversity of correlation effects (such as the induction of positive or negative correlations) caused by cross-hybridization can be expected in theory but there are natural limitations on the ability to provide quantitative insights into such effects due to the fact that they are not directly observable. The proposed stochastic model is instrumental in studying general regularities in hybridization interaction between probe sets in microarray data. As the problem stands now, there is no compelling reason to believe that multiple targeting causes a large-scale effect on the correlation structure of Affymetrix gene expression data. Our analysis suggests that the observed long-range correlations in microarray data are of a biological nature rather than a technological flaw.

Seasonal Changes in Bacterial and Archaeal Gene Expression Patterns across Salinity Gradients in the Columbia River Coastal Margin

PubMed Central

Smith, Maria W.; Herfort, Lydie; Tyrol, Kaitlin; Suciu, Dominic; Campbell, Victoria; Crump, Byron C.; Peterson, Tawnya D.; Zuber, Peter; Baptista, Antonio M.; Simon, Holly M.

2010-01-01

Through their metabolic activities, microbial populations mediate the impact of high gradient regions on ecological function and productivity of the highly dynamic Columbia River coastal margin (CRCM). A 2226-probe oligonucleotide DNA microarray was developed to investigate expression patterns for microbial genes involved in nitrogen and carbon metabolism in the CRCM. Initial experiments with the environmental microarrays were directed toward validation of the platform and yielded high reproducibility in multiple tests. Bioinformatic and experimental validation also indicated that >85% of the microarray probes were specific for their corresponding target genes and for a few homologs within the same microbial family. The validated probe set was used to query gene expression responses by microbial assemblages to environmental variability. Sixty-four samples from the river, estuary, plume, and adjacent ocean were collected in different seasons and analyzed to correlate the measured variability in chemical, physical and biological water parameters to differences in global gene expression profiles. The method produced robust seasonal profiles corresponding to pre-freshet spring (April) and late summer (August). Overall relative gene expression was high in both seasons and was consistent with high microbial abundance measured by total RNA, heterotrophic bacterial production, and chlorophyll a. Both seasonal patterns involved large numbers of genes that were highly expressed relative to background, yet each produced very different gene expression profiles. April patterns revealed high differential gene expression in the coastal margin samples (estuary, plume and adjacent ocean) relative to freshwater, while little differential gene expression was observed along the river-to-ocean transition in August. Microbial gene expression profiles appeared to relate, in part, to seasonal differences in nutrient availability and potential resource competition. Furthermore, our results suggest that highly-active particle-attached microbiota in the Columbia River water column may perform dissimilatory nitrate reduction (both dentrification and DNRA) within anoxic particle microniches. PMID:20967204

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

PubMed Central

Ramsey, John S; Wilson, Alex CC; de Vos, Martin; Sun, Qi; Tamborindeguy, Cecilia; Winfield, Agnese; Malloch, Gaynor; Smith, Dawn M; Fenton, Brian; Gray, Stewart M; Jander, Georg

2007-01-01

Background The green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. Results Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. Conclusion New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects. PMID:18021414

Functional Genomic Analysis of the Impact of Camelina (Camelina sativa) Meal on Atlantic Salmon (Salmo salar) Distal Intestine Gene Expression and Physiology.

PubMed

Brown, Tyler D; Hori, Tiago S; Xue, Xi; Ye, Chang Lin; Anderson, Derek M; Rise, Matthew L

2016-06-01

The inclusion of plant meals in diets of farmed Atlantic salmon can elicit inflammatory responses in the distal intestine (DI). For the present work, fish were fed a standard fish meal (FM) diet or a diet with partial replacement of FM with solvent-extracted camelina meal (CM) (8, 16, or 24 % CM inclusion) during a 16-week feeding trial. A significant decrease in growth performance was seen in fish fed all CM inclusion diets (Hixson et al. in Aquacult Nutr 22:615-630, 2016). A 4x44K oligonucleotide microarray experiment was carried out and significance analysis of microarrays (SAM) and rank products (RP) methods were used to identify differentially expressed genes between the DIs of fish fed the 24 % CM diet and those fed the FM diet. Twelve features representing six known transcripts and two unknowns were identified as CM responsive by both SAM and RP. The six known transcripts (including thioredoxin and ependymin), in addition to tgfb, mmp13, and GILT, were studied using qPCR with RNA templates from all four experimental diet groups. All six microarray-identified genes were confirmed to be CM responsive, as was tgfb and mmp13. Histopathological analyses identified signs of inflammation in the DI of salmon fed CM-containing diets, including lamina propria and sub-epithelial mucosa thickening, infiltration of eosinophilic granule cells, increased goblet cells and decreased enterocyte vacuolization. All of these were significantly altered in 24 % CM compared to all other diets, with the latter two also altered in 16 % CM compared with 8 % CM and control diet groups. Significant correlation was seen between histological parameters as well as between five of the qPCR analyzed genes and histological parameters. These molecular biomarkers of inflammation arising from long-term dietary CM exposure will be useful in the development of CM-containing diets that do not have deleterious effects on salmon growth or physiology.

THE PATH FROM MOLECULAR INDICATORS OF EXPOSURE TO DESCRIBING DYNAMIC BIOLOGICAL SYSTEMS IN AN AQUATIC ORGANISM: MICROARRAYS AND THE FATHEAD MINNOW

EPA Science Inventory

The extent to which humans and wildlife are exposed to toxicants is an important focus of environmental research. This work has been directed toward the development of molecular indicators diagnostic for exposure to various stressors in freshwater fish. Research includes the di...

Application of Microarrays and qPCR to Identify Phylogenetic and Functional Biomarkers Diagnostic of Microbial Communities that Biodegrade Chlorinated Solvents to Ethene

DTIC Science & Technology

2012-01-01

Acidobacteria, Actinobacteria , Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, and all classes of Proteobacteria accounted for 76% of this dynamic...the course of treatment (Figure 4). The dominant members in cluster group 2 were from Actinobacteria (24 subfamilies), Bacteroidetes (25 subfamilies

[Application of chromosomal microarray analysis for the diagnosis of children with intellectual disability/developmental delay and a normal karytype].

PubMed

Hu, Ting; Zhu, Hongmei; Zhang, Zhu; Wang, Jiamin; Liu, Hongqian; Zhang, Xuemei; Zhang, Haixia; Du, Ze; Li, Lingping; Wang, He; Liu, Shanling

2017-04-10

To assess the value of chromosomal microarray analysis (CMA) for the diagnosis of children with intellectual disability/developmental delay (ID/DD) but a normal karytype. Peripheral blood samples from 92 ID/DD patients were analyzed with CMA using Affymetrix CytoScan 750K arrays. The results were analyzed by ChAS v3.0 software. Eighteen cases (19.57%) were detected with abnormalities by CMA, among which 10 cases were diagnosed with microdeletion/microduplication syndromes. These included 2 Williams-Beuren syndromes, 2 Angelman syndromes, 2 Russell-Silver syndromes, 1 Smith-Magenis syndromes, 1 Wolf-Hirschhorn syndromes, 1 15q26 overgrowth syndrome and 1 Xq28 (MECP2) duplication syndrome. In addition, 8 cases were diagnosed with pathogenic copy number variations (pCNV). CMA can significantly improve the diagnostic rate for patients with ID/DD, which is of great value for the treatment of such children and guidance of reproduction for their parents. Therefore, CMA should become the first-line diagnostic test for patients with ID/DD.

Rare genomic rearrangement in a boy with Williams-Beuren syndrome associated to XYY syndrome and intriguing behavior.

PubMed

Dutra, Roberta L; Piazzon, Flavia B; Zanardo, Évelin A; Costa, Thais Virginia Moura Machado; Montenegro, Marília M; Novo-Filho, Gil M; Dias, Alexandre T; Nascimento, Amom M; Kim, Chong Ae; Kulikowski, Leslie D

2015-12-01

Williams-Beuren syndrome (WBS) is caused by a hemizygous contiguous gene microdeletion of 1.55-1.84 Mb at 7q11.23 region. Approximately, 28 genes have been shown to contribute to classical phenotype of SWB with presence of dysmorphic facial features, supravalvular aortic stenosis (SVAS), intellectual disability, and overfriendliness. With the use of Microarray-based comparative genomic hybridization and other molecular cytogenetic techniques, is possible define with more accuracy partial or atypical deletion and refine the genotype-phenotype correlation. Here, we report on a rare genomic structural rearrangement in a boy with atypical deletion in 7q11.23 and XYY syndrome with characteristic clinical signs, but not sufficient for the diagnosis of WBS. Cytogenetic analysis of G-banding showed a karyotype 47,XYY. Analysis of DNA with the technique of MLPA (Multiplex Ligation-dependent Probe Amplification) using kits a combination of kits (P064, P036, P070, and P029) identified an atypical deletion on 7q11.23. In addition, high resolution SNP Oligonucleotide Microarray Analysis (SNP-array) confirmed the alterations found by MLPA and revealed others pathogenic CNVs, in the chromosomes 7 and X. The present report demonstrates an association not yet described in literature, between Williams-Beuren syndrome and 47,XYY. The identification of atypical deletion in 7q11.23 concomitant to additional pathogenic CNVs in others genomic regions allows a better comprehension of clinical consequences of atypical genomic rearrangements. © 2015 Wiley Periodicals, Inc.

Signaling Pathways Mediating the Induction of Apple Fruitlet Abscission1[C][W][OA

PubMed Central

Botton, Alessandro; Eccher, Giulia; Forcato, Claudio; Ferrarini, Alberto; Begheldo, Maura; Zermiani, Monica; Moscatello, Stefano; Battistelli, Alberto; Velasco, Riccardo; Ruperti, Benedetto; Ramina, Angelo

2011-01-01

Apple (Malus × domestica) represents an interesting model tree crop for studying fruit abscission. The physiological fruitlet drop occurring in this species can be easily magnified by using thinning chemicals, such as benzyladenine (BA), to obtain fruits with improved quality and marketability. Despite the economic importance of this process, the molecular determinants of apple fruitlet abscission are still unknown. In this research, BA was used to obtain fruitlet populations with different abscission potentials to be analyzed by means of a newly released 30K oligonucleotide microarray. RNAs were extracted from cortex and seed of apple fruitlets sampled over a 4-d time course, during which BA triggers fruit drop, and used for microarray hybridization. Transcriptomic profiles of persisting and abscising fruitlets were tested for statistical association with abscission potential, allowing us to identify molecular signatures strictly related to fruit destiny. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via reactive oxygen species/sugar and hormones signaling cross talk, and followed by a block of embryogenesis and the consequent activation of the abscission zone. PMID:21037112

Protective effect of bicyclol against bile duct ligation-induced hepatic fibrosis in rats.

PubMed

Zhen, Yong-Zhan; Li, Na-Ren; He, Hong-Wei; Zhao, Shuang-Shuang; Zhang, Guang-Ling; Hao, Xiao-Fang; Shao, Rong-Guang

2015-06-21

To evaluate the protective effect of bicyclol against bile duct ligation (BDL)-induced hepatic fibrosis in rats. Sprague-Dawley male rats underwent BDL and sham-operated animals were used as healthy controls. The BDL rats were divided into two groups which received sterilized PBS or bicyclol (100 mg/kg per day) orally for two consecutive weeks. Serum, urine and bile were collected for biochemical determinations. Liver tissues were collected for histological analysis and a whole genome oligonucleotide microarray assay. Reverse transcription-polymerase chain reaction and Western blotting were used to verify the expression of liver fibrosis-related genes. Treatment with bicyclol significantly reduced liver fibrosis and bile duct proliferation after BDL. The levels of alanine aminotransferase (127.7 ± 72.3 vs 230.4 ± 69.6, P < 0.05) and aspartate aminotransferase (696.8 ± 232.6 vs 1032.6 ± 165.8, P < 0.05) were also decreased by treatment with bicyclol in comparison to PBS. The expression changes of 45 fibrogenic genes and several fibrogenesis-related pathways were reversed by bicyclol in the microarray assay. Bicyclol significantly reduced liver mRNA and/or protein expression levels of collagen 1a1, matrix metalloproteinase 2, tumor necrosis factor, tissue inhibitors of metalloproteinases 2, transforming growth factor-β1 and α-smooth muscle actin. Bicyclol significantly attenuates BDL-induced liver fibrosis by reversing fibrogenic gene expression. These findings suggest that bicyclol might be an effective anti-fibrotic drug for the treatment of cholestatic liver disease.

Alteration of Gene Expression Profile in Kidney of Spontaneously Hypertensive Rats Treated with Protein Hydrolysate of Blue Mussel (Mytilus edulis) by DNA Microarray Analysis

PubMed Central

Feng, Junli; Dai, Zhiyuan; Zhang, Yanping; Meng, Lu; Ye, Jian; Ma, Xuting

2015-01-01

Marine organisms are rich sources of bioactive components, which are often reported to have antihypertensive effects. However, the underlying mechanisms have yet to be fully identified. The aim of this study was to investigate the antihypertensive effect of enzymatic hydrolysis of blue mussel protein (HBMP) in rats. Peptides with in vitro ACE inhibitory activity were purified from HBMP by ultrafiltration, gel filtration chromatography and reversed-phase high performance liquid chromatography. And the amino acid sequences of isolated peptides were estimated to be Val-Trp, Leu-Gly-Trp, and Met-Val-Trp-Thr. To study its in vivo action, spontaneously hypertensive rats (SHRs) were orally administration with high- or low-dose of HBMP for 28 days. Major components of the renin-angiotensin (RAS) system in serum of SHRs from different groups were analyzed, and gene expression profiling were performed in the kidney of SHRs, using the Whole Rat Genome Oligonucleotide Microarray. Results indicated although genes involved in RAS system were not significantly altered, those related to blood coagulation system, cytokine and growth factor, and fatty acids metabolism were remarkablely changed. Several genes which were seldom reported to be implicated in pathogenesis of hypertension also showed significant expression alterations after oral administration of HBMP. These data provided valuable information for our understanding of the molecular mechanisms that underlie the potential antihypertensive activities of HBMP, and will contribute towards increased value-added utilization of blue mussel protein. PMID:26517713

Transcriptional profiling of Epstein–Barr virus (EBV) genes and host cellular genes in nasal NK/T-cell lymphoma and chronic active EBV infection

PubMed Central

Zhang, Y; Ohyashiki, J H; Takaku, T; Shimizu, N; Ohyashiki, K

2006-01-01

Nasal NK/T-cell lymphoma is an aggressive subtype of non-Hodgkin lymphoma (NHL) that is closely associated with Epstein–Barr virus (EBV). The clonal expansion of EBV-infected NK or T cells is also seen in patients with chronic active EBV (CAEBV) infection, suggesting that two diseases might share a partially similar mechanism by which EBV affects host cellular gene expression. To understand the pathogenesis of EBV-associated NK/T-cell lymphoproliferative disorders (LPD) and design new therapies, we employed a novel EBV DNA microarray to compare patterns of EBV expression in six cell lines established from EBV-associated NK/T-cell LPD. We found that expression of BZLF1, which encodes the immediate-early gene product Zta, was expressed in SNK/T cells and the expression levels were preferentially high in cell lines from CAEBV infection. We also analyzsd the gene expression patterns of host cellular genes using a human oligonucleotide DNA microarray. We identified a subset of pathogenically and clinically relevant host cellular genes, including TNFRSF10D, CDK2, HSPCA, IL12A as a common molecular biological properties of EBV-associated NK/T-cell LPD and a subset of genes, such as PDCD4 as a putative contributor for disease progression. This study describes a novel approach from the aspects of viral and host gene expression, which could identify novel therapeutic targets in EBV-associated NK/T-cell LPD. PMID:16449999

Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis

PubMed Central

Schwartz, Justin T.; Bandyopadhyay, Sarmistha; Kobayashi, Scott D.; McCracken, Jenna; Whitney, Adeline R.; DeLeo, Frank R.; Allen, Lee-Ann H.

2013-01-01

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24 h time period relative to the uninfected controls. Of ~47,000 genes analyzed, 3,435 were significantly up- or down-regulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes associated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2, and CASP8) and genes that act via the NF B pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2, and RELA, IL1B, CAST, CDK2, GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that X-linked inhibitor of apoptosis (XIAP) protein remained abundant in PMNs over 48 h of LVS infection, whereas BAX mRNA and protein were progressively down-regulated. These data strongly suggest that antiapoptotic and pro-survival mechanisms collaborate to sustain the viability of F. tularensis infected neutrophils. PMID:22986450

A customized gene expression microarray reveals that the brittle stem phenotype fs2 of barley is attributable to a retroelement in the HvCesA4 cellulose synthase gene.

PubMed

Burton, Rachel A; Ma, Gang; Baumann, Ute; Harvey, Andrew J; Shirley, Neil J; Taylor, Jillian; Pettolino, Filomena; Bacic, Antony; Beatty, Mary; Simmons, Carl R; Dhugga, Kanwarpal S; Rafalski, J Antoni; Tingey, Scott V; Fincher, Geoffrey B

2010-08-01

The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.

A Customized Gene Expression Microarray Reveals That the Brittle Stem Phenotype fs2 of Barley Is Attributable to a Retroelement in the HvCesA4 Cellulose Synthase Gene1[W][OA

PubMed Central

Burton, Rachel A.; Ma, Gang; Baumann, Ute; Harvey, Andrew J.; Shirley, Neil J.; Taylor, Jillian; Pettolino, Filomena; Bacic, Antony; Beatty, Mary; Simmons, Carl R.; Dhugga, Kanwarpal S.; Rafalski, J. Antoni; Tingey, Scott V.; Fincher, Geoffrey B.

2010-01-01

The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2. PMID:20530215

ENO1 Overexpression in Pancreatic Cancer Patients and Its Clinical and Diagnostic Significance

PubMed Central

Yin, Hang; Wang, Lei

2018-01-01

We investigated in this study the expression of ENO1 in tissues and plasma of PDAC patients to evaluate its clinicopathological and diagnostic significance. ENO1 protein expression was detected in tissue microarray of human PDAC and adjacent noncancer tissues. Electrochemiluminescence immunoassay and amplified luminescent proximity homogeneous assay (AlphaLISA) were performed to measure CA19-9 and ENO1 concentration in plasma from PDAC patients and healthy controls. We demonstrated that ENO1 overexpression is positively correlated with clinical stage, lymph node metastasis, and poor prognosis of PDAC; ENO1 may function as a hopeful candidate diagnostic marker in combination with CA19-9 in PDAC diagnosis. PMID:29483925

Detection of cystic fibrosis transmembrane conductance regulator ΔF508 gene mutation using a paper-based nucleic acid hybridization assay and a smartphone camera.

PubMed

Malhotra, Karan; Noor, M Omair; Krull, Ulrich J

2018-05-29

Diagnostic technology that makes use of paper platforms in conjunction with the ubiquitous availability of digital cameras in cellular telephones and personal assistive devices offers opportunities for development of bioassays that are cost effective and widely distributed. Assays that operate effectively in aqueous solution require further development for implementation in paper substrates, overcoming issues associated with surface interactions on a matrix that offers a large surface-to-volume ratio and constraints on convective mixing. This report presents and compares two related methods for determination of oligonucleotides that serve as indicators of cystic fibrosis, differentiating between the normal wild-type sequence, and a mutant-type sequence that has a 3-base replacement. The transduction strategy operates by selective hybridization of oligonucleotide probes that are conjugated to fluorescent quantum dots, where hybridization of target sequences causes a molecular fluorophore to approach the quantum dot and become emissive through fluorescence resonance energy transfer. Detection can rely on hybridization of a target that is labelled with Cy3 fluorophore, or in the presence of an unlabelled target when a sandwich assay format is implemented with a labelled reporter oligonucleotide. Selectivity to determine the presence of mismatched sequences involves appropriate selection of nucleotide sequences to set melt temperatures, in conjunction with control of stringency conditions using formamide as a chaotrope. It was determined that both direct and sandwich assays on paper substrates are able to distinguish between wild-type and mutant-type samples.

DNA-programmable nanoparticle crystallization.

PubMed

Park, Sung Yong; Lytton-Jean, Abigail K R; Lee, Byeongdu; Weigand, Steven; Schatz, George C; Mirkin, Chad A

2008-01-31

It was first shown more than ten years ago that DNA oligonucleotides can be attached to gold nanoparticles rationally to direct the formation of larger assemblies. Since then, oligonucleotide-functionalized nanoparticles have been developed into powerful diagnostic tools for nucleic acids and proteins, and into intracellular probes and gene regulators. In contrast, the conceptually simple yet powerful idea that functionalized nanoparticles might serve as basic building blocks that can be rationally assembled through programmable base-pairing interactions into highly ordered macroscopic materials remains poorly developed. So far, the approach has mainly resulted in polymerization, with modest control over the placement of, the periodicity in, and the distance between particles within the assembled material. That is, most of the materials obtained thus far are best classified as amorphous polymers, although a few examples of colloidal crystal formation exist. Here, we demonstrate that DNA can be used to control the crystallization of nanoparticle-oligonucleotide conjugates to the extent that different DNA sequences guide the assembly of the same type of inorganic nanoparticle into different crystalline states. We show that the choice of DNA sequences attached to the nanoparticle building blocks, the DNA linking molecules and the absence or presence of a non-bonding single-base flexor can be adjusted so that gold nanoparticles assemble into micrometre-sized face-centred-cubic or body-centred-cubic crystal structures. Our findings thus clearly demonstrate that synthetically programmable colloidal crystallization is possible, and that a single-component system can be directed to form different structures.

New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

PubMed Central

Gbaj, A; Bichenkova, EV; Walsh, L; Savage, HE; Sardarian, AR; Etchells, LL; Gulati, A; Hawisa, S; Douglas, KT

2009-01-01

The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing. PMID:21483539

Advanced surface-enhanced Raman gene probe systems and methods thereof

DOEpatents

Vo-Dinh, Tuan

2001-01-01

The subject invention is a series of methods and systems for using the Surface-Enhanced Raman (SER)-labeled Gene Probe for hybridization, detection and identification of SER-labeled hybridized target oligonucleotide material comprising the steps of immobilizing SER-labeled hybridized target oligonucleotide material on a support means, wherein the SER-labeled hybridized target oligonucleotide material comprise a SER label attached either to a target oligonucleotide of unknown sequence or to a gene probe of known sequence complementary to the target oligonucleotide sequence, the SER label is unique for the target oligonucleotide strands of a particular sequence wherein the SER-labeled oligonucleotide is hybridized to its complementary oligonucleotide strand, then the support means having the SER-labeled hybridized target oligonucleotide material adsorbed thereon is SERS activated with a SERS activating means, then the support means is analyzed.

Winnowing DNA for Rare Sequences: Highly Specific Sequence and Methylation Based Enrichment

PubMed Central

Thompson, Jason D.; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue. PMID:22355378

Aptamers: novel diagnostic and therapeutic tools for diabetes mellitus and metabolic diseases.

PubMed

Hu, Jingping; Ye, Mao; Zhou, Zhiguang

2017-03-01

Diabetes mellitus is one of the most common chronic diseases that threatens human health in worldwide populations. Despite enormous efforts invested in the study of diabetes mellitus, the development of precise diagnoses and treatments for this disease remains difficult due to the limitations of current techniques. Therefore, new methods are currently being developed. Aptamers are oligonucleotides that bind to specific target molecules and have been widely applied as diagnostic and therapeutic tools. In recent years, aptamers have been utilized in the study of diabetes mellitus and metabolic diseases. In this review, we highlight recent developments and new perspectives on aptamers in the field of diabetes mellitus and other metabolic diseases. Aptamers could potentially provide the means for efficient diagnoses and therapies against diabetes mellitus.

Exploring the Hybridization Thermodynamics of Spherical Nucleic Acids to Tailor Probes for Diagnostic and Therapeutic Applications

NASA Astrophysics Data System (ADS)

Randeria, Pratik Shailesh

Spherical nucleic acids (SNAs), three-dimensional nanoparticle conjugates composed of densely packed and highly oriented oligonucleotides around organic or inorganic nanoparticles, are an emergent class of nanostructures that show promise as single-entity agents for intracellular messenger RNA (mRNA) detection and gene regulation. SNAs exhibit superior biocompatibility and biological properties compared to linear oligonucleotides, enabling them to overcome many of the limitations of linear oligonucleotides for use in biomedical applications. However, the origins of these biologically attractive properties are not well understood. In this dissertation, the chemistry underlying one such property is studied in detail, and the findings are applied towards the rational design of more effective SNAs for diagnostic and therapeutic applications. Chapter 1 introduces the synthesis of SNAs, the unique properties that make them superior to linear nucleic acids for biomedicine, and previously studied applications of these structures. Chapter 2 focuses on quantitatively studying the impact of the chemical structure of the SNA on its ability to hybridize multiple complementary nucleic acids. This chapter lays the groundwork for understanding the factors that govern SNA hybridization thermodynamics and how to tailor SNAs to increase their binding affinity to target mRNA strands. Chapters 3 and 4 capitalize on this knowledge to engineer probes for intracellular mRNA detection and gene regulation applications. Chapter 3 reports the development of an SNA-based probe that can simultaneously report the expression level of two different mRNA transcripts in live cells and differentiate diseased cells from non-diseased cells. Chapter 4 investigates the use of topically-applied SNAs to down-regulate a critical mediator of impaired wound healing in diabetic mice to accelerate wound closure. This study represents the first topical therapeutic application of SNA nanotechnology to treat open wounds and lays the groundwork for developing SNA-based approaches for treating any skin-related disorder with a known genetic signature. Chapter 5 summarizes the key findings and conclusions and introduces future research directions. Taken together, this work demonstrates the important, and often surprising, role nanostructure plays in controlling biological properties and supports the continued development of SNAs as probes for molecular and medical biology.

[Study toward practical use of oligonucleotide therapeutics].

PubMed

Inoue, Takao; Yoshida, Tokuyuki

2014-01-01

Over the past decade, oligonucleotide-based therapeutics such as antisense oligonucleotides and small interfering RNAs (siRNAs) have been developed extensively. For example, mipomersen (Kynamro; ISIS Pharmaceuticals), which is a second-generation antisense oligonucleotide administered by subcutaneous injection, has recently been approved by the FDA for the treatment of homozygous familial hypercholesterolemia. On the other hands, methods for the evaluation of quality, efficacy and safety of oligonucleotide therapeutics have not been fully discussed. Furthermore, the regulatory guidance specific for oligonucleotide therapeutics has not been established yet. Under these circumstances, we started to collaborate with Osaka University and PMDA to discuss regulatory science focused on oligonucleotide therapeutics. Through the collaboration, we would like to propose the possible design of quality evaluation and preclinical safety-evaluation of oligonucleotide therapeutics.

[Microarrays in 236 patients with neurodevelopmental disorders and congenital abnormalities].

PubMed

Faundes, Víctor; Santa María, Lorena; Morales, Paulina; Curotto, Bianca; Alliende, María Angélica

2017-07-01

In 20% of neurodevelopmental disorders (NDD) and congenital abnormalities (CA) the cause would be a genomic imbalance detectable only by chromosomal microarrays (CMA). To analyze the results of CMA performed at the INTA Laboratory of Molecular Cytogenetics, during a period of four years in patients with NDD or CA. Retrospective study that included all CMA reports of Chilean patients. Age, sex, clinical diagnosis and origin were analyzed, as well as the characteristics of the finding. The percentage of cases diagnosed by CMA was calculated considering all patients with pathogenic (PV) or probably pathogenic variants (VLP). Finally, we studied the association between patients' characteristics and a positive CMA outcome. A total of 236 reports were analyzed. The median age was 5.41 (range 2.25-9.33) years, and 59% were men. Ninety chromosomal imbalances were found, which corresponded mainly to deletions (53.3%), with a median size of 1.662 (range 0.553-6.673) Megabases. The diagnostic rate of CMA in Chilean patients from all over the country was 19.2%. There was a close relationship between the patient's sex and the detection of VLP/VP (p = 0.034). Our diagnostic rate and the association between female sex and a higher percentage of diagnosed cases are concordant with other international studies. Therefore, CMA is a valid diagnostic tool in the Chilean population.

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins.

PubMed

Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

2007-12-21

Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

PubMed Central

Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

2007-01-01

Background Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences. PMID:18154678

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

PubMed

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

2016-04-05

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

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

PubMed

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

2009-02-01

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

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

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

PubMed

Sheng, Xiuzhen; Xu, Xiaoli; Zhan, Wenbin

2013-05-01

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

Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder.

PubMed

Tammimies, Kristiina; Marshall, Christian R; Walker, Susan; Kaur, Gaganjot; Thiruvahindrapuram, Bhooma; Lionel, Anath C; Yuen, Ryan K C; Uddin, Mohammed; Roberts, Wendy; Weksberg, Rosanna; Woodbury-Smith, Marc; Zwaigenbaum, Lonnie; Anagnostou, Evdokia; Wang, Zhuozhi; Wei, John; Howe, Jennifer L; Gazzellone, Matthew J; Lau, Lynette; Sung, Wilson W L; Whitten, Kathy; Vardy, Cathy; Crosbie, Victoria; Tsang, Brian; D'Abate, Lia; Tong, Winnie W L; Luscombe, Sandra; Doyle, Tyna; Carter, Melissa T; Szatmari, Peter; Stuckless, Susan; Merico, Daniele; Stavropoulos, Dimitri J; Scherer, Stephen W; Fernandez, Bridget A

2015-09-01

The use of genome-wide tests to provide molecular diagnosis for individuals with autism spectrum disorder (ASD) requires more study. To perform chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) in a heterogeneous group of children with ASD to determine the molecular diagnostic yield of these tests in a sample typical of a developmental pediatric clinic. The sample consisted of 258 consecutively ascertained unrelated children with ASD who underwent detailed assessments to define morphology scores based on the presence of major congenital abnormalities and minor physical anomalies. The children were recruited between 2008 and 2013 in Newfoundland and Labrador, Canada. The probands were stratified into 3 groups of increasing morphological severity: essential, equivocal, and complex (scores of 0-3, 4-5, and ≥6). All probands underwent CMA, with WES performed for 95 proband-parent trios. The overall molecular diagnostic yield for CMA and WES in a population-based ASD sample stratified in 3 phenotypic groups. Of 258 probands, 24 (9.3%, 95%CI, 6.1%-13.5%) received a molecular diagnosis from CMA and 8 of 95 (8.4%, 95%CI, 3.7%-15.9%) from WES. The yields were statistically different between the morphological groups. Among the children who underwent both CMA and WES testing, the estimated proportion with an identifiable genetic etiology was 15.8% (95%CI, 9.1%-24.7%; 15/95 children). This included 2 children who received molecular diagnoses from both tests. The combined yield was significantly higher in the complex group when compared with the essential group (pairwise comparison, P = .002). [table: see text]. Among a heterogeneous sample of children with ASD, the molecular diagnostic yields of CMA and WES were comparable, and the combined molecular diagnostic yield was higher in children with more complex morphological phenotypes in comparison with the children in the essential category. If replicated in additional populations, these findings may inform appropriate selection of molecular diagnostic testing for children affected by ASD.

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

PubMed

Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

2017-07-15

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

Artificial mismatch hybridization

DOEpatents

Guo, Zhen; Smith, Lloyd M.

1998-01-01

An improved nucleic acid hybridization process is provided which employs a modified oligonucleotide and improves the ability to discriminate a control nucleic acid target from a variant nucleic acid target containing a sequence variation. The modified probe contains at least one artificial mismatch relative to the control nucleic acid target in addition to any mismatch(es) arising from the sequence variation. The invention has direct and advantageous application to numerous existing hybridization methods, including, applications that employ, for example, the Polymerase Chain Reaction, allele-specific nucleic acid sequencing methods, and diagnostic hybridization methods.

A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus

PubMed Central

2011-01-01

Background Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. Results SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome. Conclusions The Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species. PMID:21492453

Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses

PubMed Central

Sforzini, Susanna; Arlt, Volker M.; Barranger, Audrey; Dallas, Lorna J.; Oliveri, Caterina; Aminot, Yann; Pacchioni, Beniamina; Millino, Caterina; Lanfranchi, Gerolamo; Readman, James W.; Moore, Michael N.; Viarengo, Aldo; Jha, Awadhesh N.

2017-01-01

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new ‘STressREsponse Microarray’ (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 μg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota. PMID:28651000

Aptamer-modified nanoparticles and their use in cancer diagnostics and treatment.

PubMed

Reinemann, Christine; Strehlitz, Beate

2014-01-06

Aptamers are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) oligonucleotides, which are able to bind their target with high selectivity and affinity. Owing to their multiple talents, aptamers combined with nanoparticles are nanosystems well qualified for the development of new biomedical devices for analytical, imaging, drug delivery and many other medical applications. Because of their target affinity, aptamers can direct the transport of aptamer-nanoparticle conjugates. The binding of the aptamers to the target "anchors" the nanoparticle-aptamer conjugates at their site of action. In this way, nanoparticle-based bioimaging and smart drug delivery are enabled, especially by use of systematically developed aptamers for cancer-associated biomarkers. This review article gives a brief overview of recent relevant research into aptamers and trends in their use in cancer diagnostics and therapy. A concise description of aptamers, their development and functionalities relating to nanoparticle modification is given. The main part of the article is dedicated to current developments of aptamer-modified nanoparticles and their use in cancer diagnostics and treatment.

Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver.

PubMed

Miller, Colton M; Tanowitz, Michael; Donner, Aaron J; Prakash, Thazha P; Swayze, Eric E; Harris, Edward N; Seth, Punit P

2018-06-01

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.

Improved detection rate of cytogenetic abnormalities in chronic lymphocytic leukemia and other mature B-cell neoplasms with use of CpG-oligonucleotide DSP30 and interleukin 2 stimulation.

PubMed

Shi, Min; Cipollini, Matthew J; Crowley-Bish, Patricia A; Higgins, Anne W; Yu, Hongbo; Miron, Patricia M

2013-05-01

Detection of cytogenetic abnormalities requires successful culture of the clonal population to obtain metaphase chromosomes for study, and as such, has been hampered by low mitotic indices of mature B cells in culture. Our study presents data on the improved abnormality detection rate with the use of a CpG-oligonucleotide/interleukin 2 (OL/IL-2) culture protocol for mature B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and non-CLL specimens. The increased detection rate of abnormalities, compared with unstimulated culture and traditional pokeweed mitogen culture, was statistically significant for both CLL and non-CLL neoplasms. For CLL specimens, our data also showed that for cytogenetically visible aberrations, OL/IL-2 was as, if not more, sensitive than detection with interphase fluorescence in situ hybridization (iFISH). Use of OL/IL-2 allowed a number of abnormalities to be detected, which were not covered by specific iFISH panels, especially balanced translocations. Therefore, OL/IL-2 stimulation improves diagnostic sensitivity and increases discovery rate of novel prognostic findings.

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

PubMed Central

Simmon, Keith; Karaca, Dilek; Langeland, Nina; Wiker, Harald G.

2012-01-01

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens. PMID:22278843

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

PubMed Central

Ai, Lin; Chen, Jun-Hu; Chen, Shao-Hong; Zhang, Yong-Nian; Cai, Yu-Chun; Zhu, Xing-Quan; Zhou, Xiao-Nong

2012-01-01

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening. PMID:23209851

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

NASA Astrophysics Data System (ADS)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-03-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

Diagnostic classification of cancer using DNA microarrays and artificial intelligence.

PubMed

Greer, Braden T; Khan, Javed

2004-05-01

The application of artificial intelligence (AI) to microarray data has been receiving much attention in recent years because of the possibility of automated diagnosis in the near future. Studies have been published predicting tumor type, estrogen receptor status, and prognosis using a variety of AI algorithms. The performance of intelligent computing decisions based on gene expression signatures is in some cases comparable to or better than the current clinical decision schemas. The goal of these tools is not to make clinicians obsolete, but rather to give clinicians one more tool in their armamentarium to accurately diagnose and hence better treat cancer patients. Several such applications are summarized in this chapter, and some of the common pitfalls are noted.

Using RNA as a tool to modify lipid nanoparticles

NASA Astrophysics Data System (ADS)

Wilner, Samantha E.

Lipid nanoparticles (LNPs) provide an attractive option for therapeutic applications because they can self-assemble and carry a diverse set of cargoes ranging from hydrophobic drugs to small interfering RNA (siRNA). Liposomes and micelles represent two classes of LNPs that have been developed for medicinal purposes; however, active targeting of LNPs to specific tissues and LNP stability in vivo remain significant challenges. We have exploited the structural characteristics and targeting ability of nucleic acids to address these obstacles. Specifically, we have introduced short nucleic acid targeting species, called aptamers, to the surface of stable nucleic acid lipid particles (SNALPs), a subset of liposomes used in siRNA delivery. In this manner, we have actively targeted SNALPs to cancer cells that overexpress the transferrin receptor (TfR). HeLa cells expressing enhanced green fluorescent protein (EGFP) were treated with SNALPs bearing an antiTfR aptamer (C2) that was identified in our lab. C2-conjugated SNALPs showed increased levels of uptake by cells by flow cytometry. More importantly, the enhanced uptake by C2-conjugated SNALPs translated to an increased level of gene knockdown when SNALPs were loaded with anti-EGFP siRNA or anti-Lamin NC siRNA. Expression of EGFP and Lamin NC decreased, respectively. These preliminary studies illustrate that aptamer-conjugated SNALPs can be designed to knock down both endogenous and exogenous genes in cancer cells with high specificity. We have also used nucleic acids to stabilize lipid micelles by introducing short quadruplex forming oligonucleotide sequences at the lipid headgroup. Micelle formation was confirmed via dynamic light scattering, transmission electron microscopy, and small angle X-ray scattering. Micelle stability was assessed using NMR and by a FRET-based assay in the presence of serum proteins. Quadruplex-stabilized micelles demonstrated enhanced stability suggesting that alterations to oligonucleotide headgroup interactions could be used to tune micelle stability. Therefore, we engineered stabilized micelles with an oligonucleotide extension and have shown that the addition of an antisense oligonucleotide leads to micelle destabilization and cargo release. The ability to control particle stability with antisense oligonucleotides represents a new paradigm in the design of programmed nanoscale devices, which we envision will find utility in the development of novel diagnostics and therapeutics.

Human Leukocyte Antigen Typing Using a Knowledge Base Coupled with a High-Throughput Oligonucleotide Probe Array Analysis

PubMed Central

Zhang, Guang Lan; Keskin, Derin B.; Lin, Hsin-Nan; Lin, Hong Huang; DeLuca, David S.; Leppanen, Scott; Milford, Edgar L.; Reinherz, Ellis L.; Brusic, Vladimir

2014-01-01

Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world’s populations, benefiting both global public health and personalized health care. PMID:25505899

Genes Involved in the Balance between Neuronal Survival and Death during Inflammation

PubMed Central

Glezer, Isaias; Chernomoretz, Ariel; David, Samuel; Plante, Marie-Michèle; Rivest, Serge

2007-01-01

Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS. PMID:17375196

Minimum probe length for unique identification of all open reading frames in a microbial genome

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

Sokhansanj, B A; Ng, J; Fitch, J P

2000-03-05

In this paper, we determine the minimum hybridization probe length to uniquely identify at least 95% of the open reading frame (ORF) in an organism. We analyze the whole genome sequences of 17 species, 11 bacteria, 4 archaea, and 2 eukaryotes. We also present a mathematical model for minimum probe length based on assuming that all ORFs are random, of constant length, and contain an equal distribution of bases. The model accurately predicts the minimum probe length for all species, but it incorrectly predicts that all ORFs may be uniquely identified. However, a probe length of just 9 bases ismore » adequate to identify over 95% of the ORFs for all 15 prokaryotic species we studied. Using a minimum probe length, while accepting that some ORFs may not be identified and that data will be lost due to hybridization error, may result in significant savings in microarray and oligonucleotide probe design.« less

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

Yeung, Y.-S.; Yip, C.-W.; Hon, C.-C.

We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by themore » diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.« less

Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

2005-01-01

The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

Experimental annotation of the human genome using microarray technology.

PubMed

Shoemaker, D D; Schadt, E E; Armour, C D; He, Y D; Garrett-Engele, P; McDonagh, P D; Loerch, P M; Leonardson, A; Lum, P Y; Cavet, G; Wu, L F; Altschuler, S J; Edwards, S; King, J; Tsang, J S; Schimmack, G; Schelter, J M; Koch, J; Ziman, M; Marton, M J; Li, B; Cundiff, P; Ward, T; Castle, J; Krolewski, M; Meyer, M R; Mao, M; Burchard, J; Kidd, M J; Dai, H; Phillips, J W; Linsley, P S; Stoughton, R; Scherer, S; Boguski, M S

2001-02-15

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Protein detection using biobarcodes.

PubMed

Müller, Uwe R

2006-10-01

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.

Identification of human cell responses to benzene and benzene metabolites.

PubMed

Gillis, Bruce; Gavin, Igor M; Arbieva, Zarema; King, Stephen T; Jayaraman, Sundararajan; Prabhakar, Bellur S

2007-09-01

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

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

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

Image microarrays (IMA): Digital pathology's missing tool

PubMed Central

Hipp, Jason; Cheng, Jerome; Pantanowitz, Liron; Hewitt, Stephen; Yagi, Yukako; Monaco, James; Madabhushi, Anant; Rodriguez-canales, Jaime; Hanson, Jeffrey; Roy-Chowdhuri, Sinchita; Filie, Armando C.; Feldman, Michael D.; Tomaszewski, John E.; Shih, Natalie NC.; Brodsky, Victor; Giaccone, Giuseppe; Emmert-Buck, Michael R.; Balis, Ulysses J.

2011-01-01

Introduction: The increasing availability of whole slide imaging (WSI) data sets (digital slides) from glass slides offers new opportunities for the development of computer-aided diagnostic (CAD) algorithms. With the all-digital pathology workflow that these data sets will enable in the near future, literally millions of digital slides will be generated and stored. Consequently, the field in general and pathologists, specifically, will need tools to help extract actionable information from this new and vast collective repository. Methods: To address this limitation, we designed and implemented a tool (dCORE) to enable the systematic capture of image tiles with constrained size and resolution that contain desired histopathologic features. Results: In this communication, we describe a user-friendly tool that will enable pathologists to mine digital slides archives to create image microarrays (IMAs). IMAs are to digital slides as tissue microarrays (TMAs) are to cell blocks. Thus, a single digital slide could be transformed into an array of hundreds to thousands of high quality digital images, with each containing key diagnostic morphologies and appropriate controls. Current manual digital image cut-and-paste methods that allow for the creation of a grid of images (such as an IMA) of matching resolutions are tedious. Conclusion: The ability to create IMAs representing hundreds to thousands of vetted morphologic features has numerous applications in education, proficiency testing, consensus case review, and research. Lastly, in a manner analogous to the way conventional TMA technology has significantly accelerated in situ studies of tissue specimens use of IMAs has similar potential to significantly accelerate CAD algorithm development. PMID:22200030

Artificial intelligence in hematology.

PubMed

Zini, Gina

2005-10-01

Artificial intelligence (AI) is a computer based science which aims to simulate human brain faculties using a computational system. A brief history of this new science goes from the creation of the first artificial neuron in 1943 to the first artificial neural network application to genetic algorithms. The potential for a similar technology in medicine has immediately been identified by scientists and researchers. The possibility to store and process all medical knowledge has made this technology very attractive to assist or even surpass clinicians in reaching a diagnosis. Applications of AI in medicine include devices applied to clinical diagnosis in neurology and cardiopulmonary diseases, as well as the use of expert or knowledge-based systems in routine clinical use for diagnosis, therapeutic management and for prognostic evaluation. Biological applications include genome sequencing or DNA gene expression microarrays, modeling gene networks, analysis and clustering of gene expression data, pattern recognition in DNA and proteins, protein structure prediction. In the field of hematology the first devices based on AI have been applied to the routine laboratory data management. New tools concern the differential diagnosis in specific diseases such as anemias, thalassemias and leukemias, based on neural networks trained with data from peripheral blood analysis. A revolution in cancer diagnosis, including the diagnosis of hematological malignancies, has been the introduction of the first microarray based and bioinformatic approach for molecular diagnosis: a systematic approach based on the monitoring of simultaneous expression of thousands of genes using DNA microarray, independently of previous biological knowledge, analysed using AI devices. Using gene profiling, the traditional diagnostic pathways move from clinical to molecular based diagnostic systems.

A review of current and future molecular diagnostic tests for use in the microbiology laboratory.

PubMed

Jannes, Geert; De Vos, Daniel

2006-01-01

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

A technological update of molecular diagnostics for infectious diseases

PubMed Central

Liu, Yu-Tsueng

2008-01-01

Identification of a causative pathogen is essential for the choice of treatment for most infectious diseases. Many FDA approved molecular assays; usually more sensitive and specific compared to traditional tests, have been developed in the last decade. A new trend of high throughput and multiplexing assays are emerging thanks to technological developments for the human genome sequencing project. The applications of microarray and ultra high throughput sequencing technologies for diagnostic microbiology are reviewed. The race for the $1000 genome technology by 2014 will have a profound impact in diagnosis and treatment of infectious diseases in the near future. PMID:18782035

Pentopyranosyl Oligonucleotide Systems. Part 11: Systems with Shortened Backbones: D)-beta-Ribopyranosyl-(4 yields 3 )- and (L)-alpha - Lyxopyranosyl-(4 yields 3 )-oligonucleotides

NASA Technical Reports Server (NTRS)

Wippo, Harald; Reck, Folkert; Kudick, Rene; Ramaseshan, Mahesh; Ceulemans, Griet; Bolli, Martin; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

2001-01-01

The (L)-a-lyxopyranosyl-(4'yields 3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4' yields 3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the six-bonds-per-backbone-unit rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-a-lyxopyranosyl-(4' yields 3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system s capability to cross-pair with DNA.

MALDI MS analysis of oligonucleotides: desalting by functional magnetite beads using microwave-assisted extraction.

PubMed

Chen, Wei-Yu; Chen, Yu-Chie

2007-11-01

The presence of alkali cation adductions of oligonucleotides commonly deteriorates matrix-assisted laser desorption/ionization (MALDI) mass spectra. Thus, desalting is required for oligonucleotide samples prior to MALDI MS analysis in order to prevent the mass spectra from developing poor quality. In this paper, we demonstrate a new approach to extract traces of oligonucleotides from aqueous solutions containing high concentrations of salts using microwave-assisted extraction. The C18-presenting magnetite beads, capable of absorbing microwave irradiation, are used as affinity probes for oligonucleotides with the addition of triethylammonium acetate as the counterions. This new microwave-assisted extraction approach using magnetite beads as the trapping agents and as microwave-absorbers has been demonstrated to be very effective in the selective binding of oligonucleotides from aqueous solutions. The extraction of oligonucleotides from solutions onto the C18-presenting magnetite beads takes only 30 s to enrich oligonucleotides in sufficient quantities for MALDI MS analysis. After using this desalting approach, alkali cation adductions of oligonucleotides are dramatically reduced in the MALDI mass spectra. The presence of saturated NaCl (approximately 6 M) in the oligonucleotide sample is tolerated without degrading the mass spectra. The detection limit for d(A)6 is approximately 2.8 fmol.

[Impact of Delivery Method on Antiviral Activity of Phosphodiester, Phosphorothioate, and Phosphoryl Guanidine Oligonucleotides in MDCK Cells Infected with H5N1 Bird Flu Virus].

PubMed

Levina, A S; Repkova, M N; Chelobanov, B P; Bessudnova, E V; Mazurkova, N A; Stetsenko, D A; Zarytova, V F

2017-01-01

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO2 nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO2 nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.

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

NASA Astrophysics Data System (ADS)

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

2010-04-01

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

[Genotyping of BRCA1, BRCA2 and CHEK2 germline mutations in Russian breast cancer patients using diagnostic biochips].

PubMed

Nasedkina, T V; Gromyko, O E; Emel'ianova, M A; Ignatova, E O; Kazubskaia, T P; Portnoĭ, S M; Zasedatelev, A S; Liubchenko, L N

2014-01-01

Germline mutations of BRCA1/2 genes cause the predisposition of their carriers to breast or/and ovary cancers (BC or/and OC) during the lifetime. Identification of these mutations is a basis of molecular diagnosis for BC susceptibility. Rapid genotyping technique using microarrays for identification of BRCA1 185delAG, 300T>G, 4153delA, 5382insC mutations and 4158 A>G sequence variant; BRCA2 695insT and 6174delT mutations; 1100delC mutation in CHEK2 gene was applied for 412 randomly collected breast cancer samples from the central region of European area of Russia. In 25 (6.0%) patients (6.0%) BC was associated with other tumours: OC, cervical cancer, colorectal cancer etc. BRCA1/2 and CHEK2 mutations were found in 33 (8.0%) BC patients. The most frequent mutation was BRCA1 5382insC, occurred in 16 (3.9%) BC patients, and CHEK2 1100delC, revealed in 7 (1.7%) BC patients. An application of diagnostic BC-microarray for genetic testing of BRCA1/2 and CHEK2 founder mutations has been discussed.

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

PubMed

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

2015-01-30

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

Modification of the 5' terminus of oligodeoxyribonucleotides for conjugation with ligands.

PubMed

Asseline, U; Thuong, N T

2001-08-01

Ligands can be introduced at the 5' terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5' terminus of the oligonucleotide. These are then reacted to give the ligand-oligonucleotide conjugate. This unit describes the addition of carboxylated and aminoalkylated linkers, and phosphorothioate, phosphate, and masked thiol groups to the 5' terminus of an oligonucleotide. The addition of linkers to ligands and the final reaction that produces the ligand-conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite- or H-phosphonate-mediated oligonucleotide synthesis.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

PubMed

Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

2017-10-01

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

Diseases and Molecular Diagnostics: A Step Closer to Precision Medicine.

PubMed

Dwivedi, Shailendra; Purohit, Purvi; Misra, Radhieka; Pareek, Puneet; Goel, Apul; Khattri, Sanjay; Pant, Kamlesh Kumar; Misra, Sanjeev; Sharma, Praveen

2017-10-01

The current advent of molecular technologies together with a multidisciplinary interplay of several fields led to the development of genomics, which concentrates on the detection of pathogenic events at the genome level. The structural and functional genomics approaches have now pinpointed the technical challenge in the exploration of disease-related genes and the recognition of their structural alterations or elucidation of gene function. Various promising technologies and diagnostic applications of structural genomics are currently preparing a large database of disease-genes, genetic alterations etc., by mutation scanning and DNA chip technology. Further the functional genomics also exploring the expression genetics (hybridization-, PCR- and sequence-based technologies), two-hybrid technology, next generation sequencing with Bioinformatics and computational biology. Advances in microarray "chip" technology as microarrays have allowed the parallel analysis of gene expression patterns of thousands of genes simultaneously. Sequence information collected from the genomes of many individuals is leading to the rapid discovery of single nucleotide polymorphisms or SNPs. Further advances of genetic engineering have also revolutionized immunoassay biotechnology via engineering of antibody-encoding genes and the phage display technology. The Biotechnology plays an important role in the development of diagnostic assays in response to an outbreak or critical disease response need. However, there is also need to pinpoint various obstacles and issues related to the commercialization and widespread dispersal of genetic knowledge derived from the exploitation of the biotechnology industry and the development and marketing of diagnostic services. Implementation of genetic criteria for patient selection and individual assessment of the risks and benefits of treatment emerges as a major challenge to the pharmaceutical industry. Thus this field is revolutionizing current era and further it may open new vistas in the field of disease management.

Novel potential serological prostate cancer biomarkers using CT100+ cancer antigen microarray platform in a multi-cultural South African cohort

PubMed Central

Adeola, Henry A.; Smith, Muneerah; Kaestner, Lisa; Blackburn, Jonathan M.; Zerbini, Luiz F.

2016-01-01

There is a growing need for high throughput diagnostic tools for early diagnosis and treatment monitoring of prostate cancer (PCa) in Africa. The role of cancer-testis antigens (CTAs) in PCa in men of African descent is poorly researched. Hence, we aimed to elucidate the role of 123 Tumour Associated Antigens (TAAs) using antigen microarray platform in blood samples (N = 67) from a South African PCa, Benign prostatic hyperplasia (BPH) and disease control (DC) cohort. Linear (fold-over-cutoff) and differential expression quantitation of autoantibody signal intensities were performed. Molecular signatures of candidate PCa antigen biomarkers were identified and analyzed for ethnic group variation. Potential cancer diagnostic and immunotherapeutic inferences were drawn. We identified a total of 41 potential diagnostic/therapeutic antigen biomarkers for PCa. By linear quantitation, four antigens, GAGE1, ROPN1, SPANXA1 and PRKCZ were found to have higher autoantibody titres in PCa serum as compared with BPH where MAGEB1 and PRKCZ were highly expressed. Also, p53 S15A and p53 S46A were found highly expressed in the disease control group. Statistical analysis by differential expression revealed twenty-four antigens as upregulated in PCa samples, while 11 were downregulated in comparison to BPH and DC (FDR = 0.01). FGFR2, COL6A1and CALM1 were verifiable biomarkers of PCa analysis using urinary shotgun proteomics. Functional pathway annotation of identified biomarkers revealed similar enrichment both at genomic and proteomic level and ethnic variations were observed. Cancer antigen arrays are emerging useful in potential diagnostic and immunotherapeutic antigen biomarker discovery. PMID:26885621

Comparative transcriptomics among floral organs of the basal eudicot Eschscholzia californica as reference for floral evolutionary developmental studies

PubMed Central

2010-01-01

Background Molecular genetic studies of floral development have concentrated on several core eudicots and grasses (monocots), which have canalized floral forms. Basal eudicots possess a wider range of floral morphologies than the core eudicots and grasses and can serve as an evolutionary link between core eudicots and monocots, and provide a reference for studies of other basal angiosperms. Recent advances in genomics have enabled researchers to profile gene activities during floral development, primarily in the eudicot Arabidopsis thaliana and the monocots rice and maize. However, our understanding of floral developmental processes among the basal eudicots remains limited. Results Using a recently generated expressed sequence tag (EST) set, we have designed an oligonucleotide microarray for the basal eudicot Eschscholzia californica (California poppy). We performed microarray experiments with an interwoven-loop design in order to characterize the E. californica floral transcriptome and to identify differentially expressed genes in flower buds with pre-meiotic and meiotic cells, four floral organs at pre-anthesis stages (sepals, petals, stamens and carpels), developing fruits, and leaves. Conclusions Our results provide a foundation for comparative gene expression studies between eudicots and basal angiosperms. We identified whorl-specific gene expression patterns in E. californica and examined the floral expression of several gene families. Interestingly, most E. californica homologs of Arabidopsis genes important for flower development, except for genes encoding MADS-box transcription factors, show different expression patterns between the two species. Our comparative transcriptomics study highlights the unique evolutionary position of E. californica compared with basal angiosperms and core eudicots. PMID:20950453