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‣ Avaliação genética e imunológica de famílias com deficiências nos componentes C3 ou Fator I do Sistema Complemento e a influência dessas deficiências na expressão gênica de fibroblastos de pele humana.; Genetic and immunologic evaluation of families with deficiencies on C3 or Factor I components of Complement System and influence of this deficiencies on gene expression of human skin fibroblasts.

Delcolli, Maria Isabel Mesquita Vendramini
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 29/06/2012 Português
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Avaliamos as causas moleculares da deficiência de C3 ou de FI em 07 pacientes, chegando a caracterizar a causa molecular em cinco deles. As mutações encontradas em cada um dos pacientes foram: C3def3 - C364G troca Arg102Gly; G2481C (Val807); A4956G (Val1632); FIdef2 - G693A, troca Gly162Asp; FIdef3 e -4 - C767T, troca Arg187Stop; FIdef6 e -7 - Inserção 1382DAT, códon de parada prematura 13 bases a montante. Além disso, analisamos se a ausência dessas proteínas levava a alterações na expressão gênica em fibroblastos de pele de pacientes deficientes de C3 ou de FI em relação a indivíduos normais e saudáveis através de ensaios com microarranjos de cDNA. Confirmamos uma tendência à alteração da expressão através de PCR em tempo real dos genes CHNRB1, CAV1, PSMB1, PI4K2B e MASP1 nos deficientes de C3; dos genes SPRY2, PSMA5, PGM2L1, GOLPH3 e JAKMIP3 nos deficientes de FI e o gene ETV6 nos dois grupos de pacientes. Dessa forma, podemos sugerir que deficiências das proteínas C3 e FI podem possivelmente influenciar a expressão de outros genes neste tipo de célula.; We investigated C3 and Factor I deficiency in 07 patients and could characterize the molecular basis in five of them. The mutations found were: C3def3 - C364G change Arg102Gly; G2481C (Val807); A4956G (Val1632); FIdef2 - G693A...

‣ Planejamento, gerenciamento e análise de dados de microarranjos de DNA para identificação de biomarcadores de diagnóstico e prognóstico de cânceres humanos; Planning, management and analysis of DNA microarray data aiming at discovery of biomarkers for diagnosis and prognosis of human cancers.

Simões, Ana Carolina Quirino
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 12/05/2009 Português
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Nesta tese, apresentamos nossas estratégias para desenvolver um ambiente matemático e computacional para análises em larga-escala de dados de expressão gênica obtidos pela tecnologia de microarranjos de DNA. As análises realizadas visaram principalmente à identificação de marcadores moleculares de diagnóstico e prognóstico de cânceres humanos. Apresentamos o resultado de diversas análises implementadas através do ambiente desenvolvido, as quais conduziram a implementação de uma ferramenta computacional para a anotação automática de plataformas de microarranjos de DNA e de outra ferramenta destinada ao rastreamento da análise de dados realizada em ambiente R. Programação eXtrema (eXtreme Programming, XP) foi utilizada como técnica de planejamento e gerenciamento dos projetos de análise dados de expressão gênica. Todos os conjuntos de dados foram obtidos por nossos colaboradores, utilizando-se duas diferentes plataformas de microarranjos de DNA: a primeira enriquecida em regiões não-codificantes do genoma humano, em particular regiões intrônicas, e a segunda representando regiões exônicas de genes humanos. A primeira plataforma foi utilizada para avaliação do perfil de expressão gênica em tumores de próstata e rim humanos...

‣ Estudo de candidatos a biomarcadores moleculares de prognóstico em carcinoma renal de células claras; Study of molecular biomarker candidates for prognosis in clear cell renal carcinoma

Vilella-Arias, Santiago Andrés
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 17/12/2013 Português
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O carcinoma de células renais (CCR) é o tumor mais agressivo que afeta o rim de pessoas adultas. O CCR é uma doença heterogênea, com diferentes alterações moleculares e variados patrões histológicos e clínicos que apresentam evolução diferente. Atualmente apenas variáveis anatomopatológicas clássicas são utilizadas para determinar o prognóstico dos pacientes. Utilizando uma plataforma de microarranjos de DNA, nosso grupo identificou em um trabalho anterior um conjunto de genes que se encontram diferencialmente expressos em tumores de rim. Neste estudo, nove candidatos foram selecionados para avaliação como marcadores de prognóstico no CCR. Foi confirmada a alteração na expressão dos genes ARNTL, ACTN4 e EPAS1 (p < 0,05) em amostras tumorais de CCR através de PCR em tempo real. Adicionalmente, foi observada a alteração da expressão dos genes ARNTL, EPAS1 e CASP7 em linhagens celulares imortalizadas derivadas de tumores renais, recapitulando por tanto, as alterações observadas nos tumores obtidos de pacientes. Posteriormente investigamos o padrão de expressão proteica destes candidatos por imunohistoquímica utilizando microarranjos de tecidos. Foi detectada a diminuição significativa (p < 0,05) da expressão das proteínas ACTN4...

‣ Avaliação de marcadores de prognóstico no câncer de mama e análise funcional de CIP4; Evaluation of prognostic markers in breast cancer and functional analysis of CIP4

Cerqueira, Otto Luiz Dutra
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 01/04/2014 Português
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O câncer de mama é uma doença extremamente heterogênea compreendendo diferentes subtipos moleculares que resultam em evoluções clínicas e condutas terapêuticas distintas. A maior gravidade desta patologia está associada a sua capacidade de formação de metástases Mudanças no padrão de expressão gênica têm sido associadas à manifestação do fenótipo metastático. Neste trabalho, utilizamos microarranjos de tecido (TMAs) para investigar a expressão de 8 biomarcadores candidatos (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3, ABG1) e avaliar seu potencial prognóstico em pacientes com carcinoma ductal invasivo da mama. Destes, ARPC3 PPIL1 e CIP4 mostraram associações estatisticamente significativas com a sobrevida câncer específica e/ou a probabilidade de desenvolvimento de metástases. Determinamos que a expressão aumentada de CIP4 nos tumores está associada a maior probabilidade de desenvolvimento de metástases. CIP4 é uma proteína adaptadora descrita na literatura como moduladora de migração e invasão celular e portanto selecionamos este candidato para caracterização funcional detalhada. Observamos que a expressão de CIP4 encontra-se aumentada em linhagens tumorais com características invasivas. A partir do silenciamento estável e regulado de CIP4 na linhagem metastática MDA-MB-231...

‣ Analise da expressão genica em diferentes especies de eucalipto utilizando a tecnologia de microarranjos de cDNA; Microarray cDNA gene expression analyses of different eucalyptus species

Jorge Lepikson Neto
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 17/11/2008 Português
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Com o intuito de obter informações relevantes para o melhoramento genético do eucalipto para a produção de biomassa, o presente trabalho buscou comparar a expressão dos genes relacionados com a formação e desenvolvimento da madeira em quatro diferentes espécies de eucalipto, tendo como objetivo identificar os padrões que as tornam mais aptas, bem como quais genes relacionados com determinadas características. Essa análise abre a possibilidade da identificação de genes chave que possam ser manipulados, através do melhoramento clássico ou da transgênia, para aumentar o conteúdo relativo de celulose das plantas, incrementando a sua eficiência para processos econômicos. 384 ESTs do banco de dados do Consórcio Genolyptus foram selecionadas para serem analisadas através da tecnologia de microarranjos de cDNA. Foram selecionadas ESTs de genes com funções conhecidas relacionadas com a formação da madeira, bem como de genes relacionados com o desenvolvimento do vegetal e de genes com função ainda desconhecida. Os dados obtidos foram cruzados com a biblioteca de ESTs do Consorcio Genolyptus (Northern Eletrônico), e foram feitos PCR em tempo real para os principais genes diferenciais nos microarranjos e para os genes da via de lignina e flavonóides. Os resultados mostraram que diferentes genes estão expressos nas espécies estudadas sendo um grande número deles ainda com função desconhecida no metabolismo do eucalipto. A maioria dos genes relacionados com a formação da parede celular não apresentou perfil de expressão diferencial nos microarranjos...

‣ Selecção de variáveis em microarrays de ADN

Silva, Luís Miguel Almeida da
Fonte: Universidade do Porto. Reitoria Publicador: Universidade do Porto. Reitoria
Tipo: Dissertação Formato: application/pdf
Português
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A aplicação de um regime específico de quimioterapia, depende de um correcto diagnóstico do paciente. Actualmente, esse diagnóstico não é efectuado de uma forma sistemática e geral, necessitando da intervenção de diferentes especialistas. Com a monitorização da expressão de milhares de genes em simultâneo, a recente tecnologia dos microarrays de ADN representa um grande passo para a sistematização do diagnóstico oncológico. No entanto, esta tecnologia produz informação em que o número de variáveis (genes) excede largamente o número de observações (amostras), o que dificulta a utilização das ferramentas estatísticas habituais de classificação. Neste sentido, torna-se necessário implementar estratégias de redução da dimensão do espaço predictor. Contudo, esta redução, que equivale a seleccionar um subconjunto de genes do conjunto inicial, deve ser extremamente direccionada, pois sabe-se que apesar do seu elevado número, apenas uma pequena parte dos genes monitorizados determina o tipo de tecido. Identificar genes com potencial predictivo é um objectivo central dos estudos de microarray aplicados à classificação de tumores. A criação de mecanismos que permitam reter apenas estes genes é essencial...

‣ Comparative transcriptome analysis between original and evolved recombinant lactose-consuming Saccharomyces cerevisiae strains

Guimarães, Pedro M. R.; Le Berre, V.; Sokol, Serguei; François, J.; Teixeira, J. A.; Domingues, Lucília
Fonte: Wiley-VCH Verlag Publicador: Wiley-VCH Verlag
Tipo: Artigo de Revista Científica
Publicado em /12/2008 Português
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The engineering of Saccharomyces cerevisiae strains for lactose utilization has been attempted with the intent of developing high productivity processes for alcoholic fermentation of cheese whey. A recombinant S. cerevisiae flocculent strain that efficiently ferments lactose to ethanol was previously obtained by evolutionary engineering of an original recombinant that displayed poor lactose fermentation performance. We compared the transcriptomes of the original and the evolved recombinant strains growing in lactose, using cDNA microarrays. Microarray data revealed 173 genes whose expression levels differed more than 1.5-fold. About half of these genes were related to RNA-mediated transposition. We also found genes involved in DNA repair and recombination mechanisms, response to stress, chromatin remodeling, cell cycle control, mitosis regulation, glycolysis and alcoholic fermentation. These transcriptomic data are in agreement with some of the previously identified physiological and molecular differences between the recombinants, and point to further hypotheses to explain those differences.

‣ Selection of Escherichia coli heat shock promoters toward their application as stress probes

Rodrigues, Joana Lúcia; Sousa, M. S.; Prather, Kristala L. J.; Kluskens, Leon; Rodrigues, L. R.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2014 Português
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The mechanism of heat shock response of Escherichia coli can be explored to program novel biological functions. In this study, the strongest heat shock promoters were identified by microarray experiments conducted at different temperatures (37 °C and 45 °C, 5 min). The promoters of the genes ibpA, dnaK and fxsA were selected and validated by RT-qPCR. These promoters were used to construct and characterize stress probes using green fluorescence protein (GFP). Cellular stress levels were evaluated in experiments conducted at different shock temperatures during several exposure times. It was concluded that the strength of the promoter is not the only relevant factor in the construction of an efficient stress probe. Furthermore, it was found to be crucial to test and optimize the ribosome binding site (RBS) in order to obtain translational efficiency that balances the transcription levels previously verified by microarrays and RT-qPCR. These heat shock promoters can be used to trigger in situ gene expression of newly constructed biosynthetic pathways.

‣ Transcriptional profiling of HOXA9-regulated genes in human glioblastoma cell models

Gonçalves, Céline S.; Magalhães, Ana Xavier; Pojo, Marta; Oliveira, Ana Isabel; Reis, R. M.; Sousa, Nuno; Rocha, Miguel; Costa, Bruno Marques
Fonte: Elsevier Inc. Publicador: Elsevier Inc.
Tipo: Artigo de Revista Científica
Publicado em //2015 Português
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The data here described pertain to the article by Pojo et al. (2015) titled A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide (Pojo, Goncalves et al. 2015). HOX genes are part of the homeobox genes family, which encodes transcription factors crucial during embryonic development [6] and [9] and also in postdevelopmental regulation [8], [14], [13] and [7]. Alterations interfering with the regulation of these genes may lead to tumorigenesis in adults. Due to their contributions in the control of important cellular processes, the deregulation of HOX genes are ultimately correlated with cancer treatment failure and patients poor prognosis ( [5] and [1]; Costa, Smith et al. 2010; Pojo, Goncalves et al. 2015). Recently, our studies showed that HOXA9 overexpression is associated with poor prognosis in patients with glioblastoma (GBM), the most common and most malignant primary brain tumor. Mechanistically, HOXA9 is associated with resistance to chemotherapy and with pro-proliferative, pro-invasive and anti-apoptotic features (Costa, Smith et al. 2010; Pojo, Goncalves et al. 2015) in GBM in vitro models. Since HOXA9 is a transcription factor, its target genes can be the true biological effectors of its aggressiveness. In this context...

‣ Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine

Rocheta, M.; Becker, J.D.; Coito, J.L.; Carvalho, L.; Amâncio, S.
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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Grapevine is an extremely important crop worldwide. In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress (WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However...

‣ Chromosomal microarrays testing in children with developmental disabilities and congenital anomalies

Lay-Son,Guillermo; Espinoza,Karena; Vial,Cecilia; Rivera,Juan C.; Guzmán,María L.; Repetto,Gabriela M.
Fonte: Sociedade Brasileira de Pediatria Publicador: Sociedade Brasileira de Pediatria
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2015 Português
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OBJECTIVES: Clinical use of microarray-based techniques for the analysis of many developmental disorders has emerged during the last decade. Thus, chromosomal microarray has been positioned as a first-tier test. This study reports the first experience in a Chilean cohort. METHODS: Chilean patients with developmental disabilities and congenital anomalies were studied with a high-density microarray (CytoScan(tm) HD Array, Affymetrix, Inc., Santa Clara, CA, USA). Patients had previous cytogenetic studies with either a normal result or a poorly characterized anomaly. RESULTS: This study tested 40 patients selected by two or more criteria, including: major congenital anomalies, facial dysmorphism, developmental delay, and intellectual disability. Copy number variants (CNVs) were found in 72.5% of patients, while a pathogenic CNV was found in 25% of patients and a CNV of uncertain clinical significance was found in 2.5% of patients. CONCLUSION: Chromosomal microarray analysis is a useful and powerful tool for diagnosis of developmental diseases, by allowing accurate diagnosis, improving the diagnosis rate, and discovering new etiologies. The higher cost is a limitation for widespread use in this setting.

‣ Hunting for differentially expressed genes

Vedoy,C.G.; Bengtson,M.H.; Sogayar,M.C.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/07/1999 Português
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Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Several methods have been used for this purpose, including differential hybridization, cDNA subtraction, differential display and, more recently, DNA chips. Subtractive hybridization has significantly improved after the polymerase chain reaction was incorporated into the original method and many new protocols have been established. Recently, the availability of the well-known coding sequences for some organisms has greatly facilitated gene expression analysis using high-density microarrays. Here, we describe some of these modifications and discuss the benefits and drawbacks of the various methods corresponding to the main advances in this field.

‣ Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection

Bellodi-Privato,M.; Kubrusly,M.S.; Stefano,J.T.; Soares,I.C.; Wakamatsu,A.; Oliveira,A.C.; Alves,V.A.F.; Bacchella,T.; Machado,M.C.C.; D’Albuquerque,L.A.C.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2009 Português
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Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher’s linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKβ, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios...

‣ Analysis of topoisomerase function in bacterial replication fork movement: Use of DNA microarrays

Khodursky, Arkady B.; Peter, Brian J.; Schmid, Molly B.; DeRisi, Joseph; Botstein, David; Brown, Patrick O.; Cozzarelli, Nicholas R.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 15/08/2000 Português
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We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.

‣ cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene

Khan, Javed; Bittner, Michael L.; Saal, Lao H.; Teichmann, Ulrike; Azorsa, David O.; Gooden, Gerald C.; Pavan, William J.; Trent, Jeffrey M.; Meltzer, Paul S.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 09/11/1999 Português
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Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

‣ Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Diehl, Frank; Grahlmann, Susanne; Beier, Markus; Hoheisel, Jörg D.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 01/04/2001 Português
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Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-l-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.

‣ An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression

Yue, Huibin; Eastman, P. Scott; Wang, Bruce B.; Minor, James; Doctolero, Michael H.; Nuttall, Rachel L.; Stack, Robert; Becker, John W.; Montgomery, Julie R.; Vainer, Marina; Johnston, Rick
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 15/04/2001 Português
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The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12–14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

‣ Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

Mahalingam, Ramamurthy; Fedoroff, Nina
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 19/06/2001 Português
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We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1...

‣ DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

Murray, A. E.; Lies, D.; Li, G.; Nealson, K.; Zhou, J.; Tiedje, J. M.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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DNA microarrays constructed with full length ORFs from Shewanella oneidensis, MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes (arcA and ATP synthase) were identified among all species investigated. The mtr operon, which is involved in iron reduction, was poorly conserved among other known metal-reducing Shewanella species. Results were most informative for closely related organisms with small subunit rRNA sequence similarities greater than 93% and gyrB sequence similarities greater than 80%. At this level of relatedness, the similarity between hybridization profiles was strongly correlated with sequence divergence in the gyrB gene. Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly identical, with only 3% of the ORFs hybridizing poorly, in contrast to hybridizations with Shewanella putrefaciens, formerly considered to be the same species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below −0.75). Genomic hybridizations showed that genes in operons had consistent levels of hybridization across an operon in comparison to a randomly sampled data set...

‣ Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries

Lindroos, Katarina; Liljedahl, Ulrika; Raitio, Mirja; Syvänen, Ann-Christine
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/2001 Português
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In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.