DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.; DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados...

Análise de expressão gênica em larga escala é de fundamental importância para a biologia molecular atual pois possibilita a medida dos níveis de expressão de milhares de genes simultaneamente, o que torna viável a realização de trabalhos voltados para biologia de sistemas (systems biology). Dentre as principais técnicas experimentais disponíveis para esta finalidade, a tecnologia de microarray tem sido amplamente utilizada. Este procedimento para medida de expressão gênica é bastante complexo e os dados obtidos são freqüentemente observacionais, o que dificulta a modelagem estatística. Não existe um protocolo padrão para a geração e avaliação desses dados, sendo portanto necessário buscar procedimentos de análise que sejam adequados para cada caso. Assim, os principais métodos matemáticos e estatísticos aplicados para a análise desses dados deveriam estar disponíveis de uma forma organizada, coerente e simples em um ambiente computacional que confira robustez, confiabilidade e reprodutibilidade às análises realizadas. Uma forma de garantir estas características é através da representação (e documentação) de todos os algoritmos utilizados na forma de um grafo direcionado e acíclico que descreva todo o conjunto de transformações...

Acredita-se que a placa dento-bacteriana seja primordial na iniciação do processo de inflamação periodontal. Entretanto, os mecanismos exatos responsáveis pela progressão da doença periodontal ainda não foram completamente elucidados. Além disso, as bases biológicas que ditam a susceptibilidade do hospedeiro ainda permanecem desconhecidas. Portanto, o presente estudo procurou investigar diferenças na expressão gênica de sítios com periodontite. Para tanto, foi utilizada a tecnologia de microarray focado para examinar a expressão de 288 genes relacionados à apoptose, resposta de células T e inflamação. RNA total foi extraído de amostras de tecido gengival e sangue de indivíduos com periodontite crônica (N=10) e de indivíduos periodontalmente saudáveis (N=4). RNA total foi então convertido em cRNA, marcado e hibridizado em lâminas de microarray. Após a exposição, os genes marcados foram quantificados e o nível de expressão genética nas amostras gengivais dos pacientes periodontais foi comparado com o nível de expressão observado no sangue desses mesmos indivíduos, e também com o nível da expressão genética no tecido gengival dos indivíduos sem doença periodontal. Para validação dos resultados dos genes selecionados (p< ou = 0...

O extraordinário potencial regenerativo do complexo dentino-pulpar enfatiza a importância da caracterização dos processos celulares e moleculares envolvidos na regeneração dentinária. O avanço da pesquisa com células-tronco desencadeou um grande interesse de cultivá-las na presença de sinais de indução odontogênica. O objetivo do presente trabalho foi avaliar comparativamente células indiferenciadas da polpa (OD-21) e odontoblásticas (MDPC-23) através da avaliação do estímulo celular e do perfil de expressão gênica. As células OD-21 e MDPC-23 foram cultivadas em garrafas de cultura até a subconfluência e, em seguida, cultivadas em placas de 24 poços na concentração de 104 células /poço (n = 5). Os parâmetros analisados foram: (1) proliferação, viabilidade celular e atividade de fosfatase alcalina após 3, 7 e 10 dias; além de detecção e quantificação de matriz mineralizada após 17 dias (o teste estatístico utilizado foi o de Mann-Whitney para p≤0,05); (2) imunofluorescência para proteínas não-colágenas (DSPP e osteopontina) após 1, 3 e 7 dias; (3) análises de expressão transcricional através da tecnologia de cDNA microarray e PCR em tempo real. Os dados de microarrays foram analisados com o auxílio de programas de bioinformática especializados como SAM (significance analysis of microarrays)...

(Microarray technology in study of head neck cancer). The microarray technology is a tool for global analysis of gene expression that allows investigating hundreds or thousands of genes in a sample using a hybridization reaction. This technology is based on hybridization between labeled targets derived from biological samples and an array of many DNA probes immobilized on a solid matrix, representing the genes of interest. The simultaneous study of hundreds of genes became the microarray technique a very important tool of global analysis, with applications in several areas, including the study of the development of cancer. Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide, with a global annual incidence of 780,000 new cases. Large-scale studies involving microarrays have identified specific gene expression signatures associated with expression changes in HNSCC samples compared to normal tissue, as well as genes involved in clinical outcome and metastasis. However, the considerable heterogeneity among these studies occurs due to experimental design, number of samples, disease sites and stage, choice of microarray platform and results validation. Thus, there is much to be validated, before the technique has clinical utility. In relation to head and neck neoplasia...

Research Article; Background: Quantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes. Results: Using the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination...

The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix). Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus) submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner). In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core.

The rapid technological development of high-throughput genomics has given rise to complex high-dimensional microarray datasets. One strategy for reducing the dimensionality of microarray experiments is to carry out a cluster analysis to find groups of genes with similar expression patterns. Though cluster analysis has been studied extensively, the clinical context in which the analysis is performed is usually considered separately if at all. However, allowing clinical outcomes to inform the clustering of microarray data has the potential to identify gene clusters that are more useful for describing the clinical course of disease. The aim of this dissertation is to utilize outcome information to drive the clustering of gene expression data. In Chapter 1, we propose a joint clustering model that assumes a relationship between gene clusters and a continuous patient outcome. Gene expression is modeled using cluster specific random effects such that genes in the same cluster are correlated. A linear combination of these random effects is then used to describe the continuous clinical outcome. We implement a Markov chain Monte Carlo algorithm to iteratively sample the unknown parameters and determine the cluster pattern. Chapter 2 extends this model to binary and failure time outcomes. Our strategy is to augment the data with a latent continuous representation of the outcome and specify that the risk of the event depends on the latent variable. Once the latent variable is sampled...

Reductionist biology has yielded tremendous insight into the basis of biochemistry and genetic disease. However, the remarkable failure of reductionist biology to explain complex problems, especially cancer, has led to the development of systems biology. The vast complexity of biological systems remains the most difficult problem in biology today. In order to understand this complexity, we need tools to massively multiplex measurements of a signaling network. Therefore, we developed lysate microarray technology to fill this need. In this work, we discuss three ways in which lysate microarrays were applied to human disease. In the first work, we discuss a key stage in malaria development. The liver-stage malaria parasite represents a promising target for intervention, and we present the first use of lysate microarray technology as a screening tool for host-parasite interactions in an infectious disease. We identified three cancer-related pathways that are modified in malaria infection, and studied the p53 pathway in depth. Our finding that the parasite downregulates p53 and that treatment with Nutlin-3 strongly decreases parasite load may lead to the development of a prophylactic malaria vaccine. In the second work, we began by screening drug combinations and varying dosing schedule in triple-negative breast cancers (TNBCs). We systematically explored stimulation space and collected a large lysate microarray dataset...

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biostatistics and Computational Biology, 2008.; Microarray technology has become an indispensable tool as it can measure expression levels of thousands of genes simultaneously. Gene expression microarrays are widely used to construct gene networks and to nd genes that are di erentially expressed between two (e.g., treatment versus control) or more phenotypes. The most basic issue to be addressed in this setting is that of multiple hypothesis testing. Numerous books and papers have discussed this issue since the advent of microarray technology more than ten years ago. However, most currently practiced methods of signi cance testing in microarray gene expression pro ling remain unstable, and the variation of the number of false discoveries is high for typical situations with A ymetrix technology. These undesirable properties are due to the fact that the number of tests is typically orders of magnitude larger than the available sample size, as well as to the presence of strong and long-range (involving thousands of genes) correlations between gene expression levels, the latter having been well documented in the literature and in our own studies. This dissertation is focused on possible causes of between-gene dependencies and their e ects on the performance of gene selection procedures. It is commonly believed that the typically high correlations in gene pairs are attributable to technological aws. This view...

Osteoarthritis (OA) is characterized by alterations to subchondral bone as well as articular cartilage. Changes to bone in OA have also been identified at sites distal to the affected joint, which include increased bone volume fraction and reduced bone mineralization. Altered bone remodelling has been proposed to underlie these bone changes in OA. To investigate the molecular basis for these changes, we performed microarray gene expression profiling of bone obtained at autopsy from individuals with no evidence of joint disease (control) and from individuals undergoing joint replacement surgery for either degenerative hip OA, or fractured neck of femur (osteoporosis [OP]). The OP sample set was included because an inverse association, with respect to bone density, has been observed between OA and the low bone density disease OP. Compugen human 19K-oligo microarray slides were used to compare the gene expression profiles of OA, control and OP bone samples. Four sets of samples were analyzed, comprising 10 OA-control female, 10 OA-control male, 10 OA-OP female and 9 OP-control female sample pairs. Print tip Lowess normalization and Bayesian statistical analyses were carried out using linear models for microarray analysis, which identified 150 differentially expressed genes in OA bone with t scores above 4. Twenty-five of these genes were then confirmed to be differentially expressed (P < 0.01) by real-time PCR analysis. A substantial number of the top-ranking differentially expressed genes identified in OA bone are known to play roles in osteoblasts...

Stress kann in verschiedenen Varianten auftreten, und sich sehr unterschiedlich auf den Organismus auswirken. Im Rahmen dieses Forschungsprojektes wurde ein freiwillig auferlegter physischer Stress in Form von Hochleistungssport untersucht. Bei hohen Trainings- oder Wettkampfbelastungen werden Stressantworten im Immunsystem des Sportlers mobilisiert, die dann sekundäre Auswirkungen auf somatische Parameter zeigen. Diese Auswirkungen können zu Veränderungen in der zellulären Zusammensetzung im peripheren Blut führen. Des Weiteren, kann man eine Veränderung auf Genexpressionsebene beobachten. Im Rahmen der Dissertationsarbeit wurden home-made cDNA Microarrays hergestellt und etabliert. Die Herstellung und Etablierung sind im Abschnitt Methode dieser Arbeit ausführlich beschrieben. In dieser Studie wurde mit home-made cDNA Microarrays die Genexpression bei acht Halbmarathonläufern untersucht. Der eingesetzte cDNA Microarray enthielt ausgewählte Gene des Stress- und Entzündungsgeschehen. Das Besondere dieses Ansatzes ist, dass nicht nur ein einzelnes oder wenige Kandidatengene sondern eine größere Gruppe potentiell relevanter Gene auf mRNA- Ebene analysiert wurde. Die Abnahmezeitpunkte der Blutproben der Läufer waren, vor (t0)...

Die Posttraumatische Belastungsstörung (PTSD) könnte sich im Zuge einer Fehladaptation des Körpers zur Abwehr der akuten Stressantwort entwickeln. Aufgrund des sympatischen Hyperarousals in PTSD wurde schon früh angenommen, dass bei Patienten mit Posttraumatischen Belastungsstörungen das Immunsystem betroffen sein könnte. Mit selbst hergestellten cDNA Microarrays, die vornehmlich Gene aus Stressgeschehen, Inflammation sowie Apoptose enthielten, wurden in der vorliegenden Arbeit Auswirkungen von PTSD auf Genexpressionsebene untersucht. Erhebliche Anstrengungen wurden unternommen, um sämtliche Bedingungen, die die Messungen beeinträchtigen könnten, zu standardisieren. Besonders erwähnenswert ist, dass es sich beim untersuchten Patientenkollektiv um eine homogene Gruppe handelt, da alle Probanden das selbe traumatische Erlebnis hatten (Flugkatatstrophe von Ramstein, 1989). Trotz des langen Zeitintervalls zwischen traumatischem Erlebnis und dieser Studie zeigten sämtliche Probanden immer noch typische Auffälligkeiten bei den diagnostischen Interviews, sowie typische psychosomatische Symptome. Das Studienkollektiv umfasste acht Patienten und acht Kontrollpersonen. Des weiteren wurde großer Aufwand betrieben, um die Microarray Versuche unter bestmöglichen standardisierten Bedingungen durchzuführen. Nach Normalisierung der Daten wurden drei statistische Methoden angewendet...

Invasive Mykosen stellen eine wachsende Gefahr, insbesondere für immunsupprimierte Patienten, dar. Dabei hängt die Prognose wesentlich von einer umgehend eingeleiteten spezifischen Therapie auf der Basis einer schnellen und präzisen Diagnose ab. Die bisherigen diagnostischen Methoden sind jedoch zeitaufwendig und die Ergebnisse oft zu unspezifisch. Der 2004 am ITB Stuttgart entwickelte Oligonukleotid-Microarray zur Detektion von humanpathogenen Pilzen (Leinberger, 2004) ist in der Lage, ausgewählte Candida- und Aspergillusspezies schnell und zuverlässig zu identifizieren. In der vorliegenden Arbeit wurde dieses System von 12 Spezies aus 2 Gruppen auf 40 Spezies aus 15 Gruppen erweitert. Ein besonderer Schwerpunkt lag dabei auf den klinisch immer bedeutsamer werdenden Zygomyceten, von denen folgende Vertreter aufgenommen wurden: Absidia corymbifera und Absidia glauca, Mucor hiemalis, Rhizomucor pusillus, und Rhizopus oryzae. Für die DNS Extraktion aus den Pilzzellen wurde ein neues Protokoll verwendet, dass die Arbeitszeit von vier auf eine Stunde verkürzte. Das Extraktionsergebnis wurde dadurch nicht beeinträchtigt: Aus allen Spezies konnte DNS in ausreichender Menge isoliert werden. Um den optimalen Versuchsaufbau zu finden...

Ziel: Die vorliegende Studie wurde durchgeführt um differentiell exprimierte Gene und Signalwege zwischen Tumor- und Kontrollgewebe zu identifizieren, die eine möglicherweise wichtige Rolle in der Krankheitsentstehung spielen. Zur Bestimmung des Expressionsprofils diente uns eine cDNA Microarray und die Auswertung der resultierenden Daten erfolgte mit der Ingenuity Pathway Analysis Software (IPA, www.ingenuity.com). Hintergrund: Vestibularisschwannome sind benigne Tumore, die von den Schwannzellen des VIII. Hirnnerven ausgehen. Mit einem Anteil von 80-86% aller raumfordernden Läsionen des Kleinhirnbrückenwinkels stellen sie somit die am häufigsten dort anzutreffende Tumorentität dar. Unter den Vestibularisschwannomen werden eine unilateral sporadische Form, eine zystische Variante und eine bilateral oder auch Neurofibromatose Typ 2 (NF2)-assoziierte Form unterschieden. Letztere ist eine charakteristische Manifestation innerhalb des autosomal-dominant vererbten Tumorsyndroms, der Neurofibromatose Typ 2 (NF2). Zudem weisen die Patienten mit einer NF2 weitere benigne Tumore des Nervensystems (z.B. Meningeome), okuläre Veränderungen (z.B. Katarakte) aber auch Hautläsionen, wie beispielsweise die typischen Café-au-lait Flecken...

Introduction: There’s a consensus to perform chromosomal microarray technique as first-tier clinical diagnostic test for individuals with developmental disabilities. However, given the complexity of clinical presentations, often several diagnostic methods are held before conducting microarray. Method: We report the case of a 5 year-old boy referred to Medical Genetics due to short stature, developmental disabilities and facial dysmorphic features. He was born from eutocic delivery after an uneventful pregnancy. He had psychomotor milestones delayed like sitting at 9 months and walking at 24 months, holding an immature broad-based gait. There was history of learning difficulties from both parents, and the mother has also short stature. On examination it was noted some facial dysmorphic features like high forehead, conical canines and rarefaction of the distal portion of the eyebrows. Due to the history of an episode of transient ataxia, and suspicion of an inherited metabolic disorder, he had already performed various analytical and imaging screenings, all normal. Results: Chromosomal microarray analysis revealed two pathogenic Copy Number Variants (CNV’s): 16p11.2 deletion and 15q11.2q13.1 duplication. The 15q11q13 microduplication syndrome (OMIM # 608636) is a very rare clinical entity with about 30 reported cases with maternal origin...

Article available at http://dx.doi.org/10.1186/1471-2164-7-117; [Background] The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. Antigenic variants of FMDV have been classically diagnosed by immunological or RT-PCR-based methods. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Recently, a FMDV microarray was described to detect simultaneously the seven FMDV serotypes. These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen.; [Results] A FMDV microarray was designed and optimized to detect SNPs at a major antigenic site of the virus. A screening of point mutants of the genomic region encoding antigenic site A of FMDV C-S8c1 was achieved. The hybridization pattern of a mutant includes specific positive and negative signals as well as crosshybridization signals...

Els experiments DNA microarray generen milers de dades en forma d'imatges al voltant de tot món. Aquesta informació ha de ser eficientment emmagatzemada ja que cal guardar fins que les tècniques d'investigació estiguin cent per cent desenvolupades i estandarditzades. A partir de l'estudi de les característiques de les imatges DNA microarray, aquest article descriu un algorisme de compressió d'imatge sense pèrdua basat en la autosimilaridad de les mateixes imatges. S'han generat taules de dades a partir d'experimentar amb la codificació definida en aquest article. Dels resultats obtinguts s'observa que en determinats casos s'aconsegueix obtenir una millora de fins a gairebé 2 bpp a la compressió d'una imatge codificada respecte a una sense codificar pel algoritme basat en la autosimilaridad. En altres casos aquesta codificació significa fins a 3 bpp d'empitjorament en els resultats. En conclusió, amb una tècnica encara per millorar s'ha demostrat que utilitzant la autosimilaridad de les imatges DNA microarray per fer una codificació és possible obtenir millors resultats de compressió.; Los experimentos DNA microarray generan miles de datos en forma de imágenes alrededor de todo mundo. Esta información tiene que ser eficientemente almacenada ya que es necesario guardarla hasta que las técnicas de investigación estén cien por ciento desarrolladas y estandarizadas. A partir del estudio de las características de las imágenes DNA microarray...

DNA microarrays are one of the fastest-growing new technologies in the field of genetic research, and DNA microarray images continue to grow in number and size. Since analysis techniques are under active and ongoing development, storage, transmission and sharing of DNA microarray images need be addressed, with compression playing a significant role. However, existing lossless coding algorithms yield only limited compression performance (compression ratios below 2:1), whereas lossy coding methods may introduce unacceptable distortions in the analysis process. This work introduces a novel Relative Quantizer (RQ), which employs non-uniform quantization intervals designed for improved compression while bounding the impact on the DNA microarray analysis. This quantizer constrains the maximum relative error introduced into quantized imagery, devoting higher precision to pixels critical to the analysis process. For suitable parameter choices, the resulting variations in the DNA microarray analysis are less than half of those inherent to the experimental variability. Experimental results reveal that appropriate analysis can still be performed for average compression ratios exceeding 4.5:1.

A problem in analyzing microarray-based gene expression data is the separation of genes causally involved in a disease from innocent bystander genes, whose expression levels have been secondarily altered by primary changes elsewhere. To investigate this issue systematically in the context of a class of complex human diseases, we have compared microarray-based gene expression data with non-microarray-based clinical and biological data about the schizophrenias to ask whether these two approaches prioritize the same genes. We find that genes whose expression changes are deemed to be of importance from microarrays are rarely those classified as of importance from clinical, in situ, molecular, single-nucleotide polymorphism (SNP) association, knockout and drug perturbation data. This disparity is not limited to the schizophrenias but characterizes other human disease data sets. It also extends to biological validation of microarray data in model organisms, in which genome-wide phenotypic data have been systematically compared with microarray data. In addition, different bioinformatic protocols applied to the same microarray data yield quite different gene sets and thus make clinical decisions less straightforward. We discuss how progress may be improved in the clinical area by the assignment of high-quality phenotypic values to each member of a microarray-assigned gene set.