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Análise comparativa dos transcritomas do córtex adrenal normal e adenocarcinoma do córtex adrenal; Comparative analysis of normal adrenal cortice and adrenocortical carcinoma transcriptomes

Gouvea, Mirian Nakamura
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 27/02/2008 PT
Relevância na Pesquisa
17.34%
A carcinogênese do córtex da supra-renal é um processo complexo que envolve alterações genéticas múltiplas e seqüenciais. Embora algumas dessas alterações já tenham sido caracterizadas, de modo geral estes mecanismos permanecem pouco compreendidos. Um conhecimento mais aprofundado dos mesmos não só levaria à descoberta de novos marcadores de prognóstico como também a alvos terapêuticos em potencial. A fim de melhor caracterizarmos os mecanismos envolvidos na progressão maligna do tumor e selecionarmos genes candidatos a serem marcadores de malignidade e alvos terapêuticos, nós estudamos os RNAs de uma linhagem celular derivada de um tumor adrenocortical maligno (NCI-H295A) e um espécime de tumor do córtex da supra-renal por "Differential Display" (DDRT-PCR). Foi selecionado um total de 317 transcritos únicos diferencialmente expressos, com base na análise densitométrica digital dos géis de DDRTPCR. A anotação funcional dos genes hiper-expressos mostrou relação com motilidade celular e proliferação. Entre os hipo-expressos foram identificados genes envolvidos na regulação de transcrição, síntese e processamento de RNA e remodelamento da cromatina. A expressão de dois genes entre os transcritos selecionados foi verificada por RT-PCR semi-quantitativa em 19 tumores adrenocorticais adultos e pediátricos...

Identificação de marcadores moleculares para o câncer de tireóide por cDNA microarrays; Identification of molecular markers for thyroid cancer by cDNA microarrays

Stolf, Beatriz Simonsen
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 29/09/2003 PT
Relevância na Pesquisa
17.34%
Doenças tireoideanas são bastante comuns, sendo sua maioria benigna. A relação entre os diversos tipos de doenças tireoideanas, bem como seus aspectos moleculares, são pouco conhecidos. O bócio (hiperplasia), por exemplo, é descrito por alguns como relacionado com carcinoma (tumor maligno) papilífero, enquanto que outros afirmam não haver relação causal entre as duas doenças. A questão mais desafiante, porém, refere-se à distinção entre adenoma (tumor benigno) e carcinoma folicular, que atualmente é feita apenas após à cirurgia, não permitindo tratamento diferenciado para os dois tipos de tumor. Este trabalho buscou identificar genes diferencialmente expressos entre tecido tireoideano normal, bócio, adenoma e carcinoma papilífero utilizando microarrays. Carcinomas foliculares não foram incluídos devido ao número e tamanho reduzidos das amostras. Dois tipos de array foram utilizados: arrays em membranas de nylon, contendo 213 clones obtidos por DDRT-PCR de amostras de tireóide, e arrays em vidro, contendo 3800 clones ORESTES. Experimentos utilizando o primeiro tipo de array identificaram três genes diferencialmente expressos, cuja expressão foi analisada por RT-PCR em 10 amostras de cada tipo de tecido. Dois deles foram capazes de diferenciar carcinomas papilíferos de tecido normal e bócio com 89% de precisão para o tumor maligno e 80% para os tecidos não malignos. Os arrays em vidro foram utilizados para avaliar o perfil de expressão de aproximadamente 10 amostras de cada tipo de tecido tireoideano. Foram identificados 160 clones diferencialmente expressos entre quaisquer dois tipos de tecido...

Efeitos do 17-estradiol e da lâmina na regulação da expressão dos genes DDEF2 e PHLDA1 em linhagens de células derivadas de adenocarcinomas de mama MCF-7 e MDA-MB-231; Transcriptional up-regulation of PHLDA1 by 17B-estradiol in MCF-7 breast cancer cells

Marchiori, Ana Carolina
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 14/04/2008 PT
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17.34%
O câncer de mama é a doença maligna mais comum e a principal causa de morte entre as mulheres. Sua complexa etiologia envolve múltiplos fatores de risco, a maioria deles relacionada aos níveis cumulativos de exposição da mama aos estrógenos. A maioria de suas ações é mediada pela ligação a seus receptores ER e ER que são fatores de transcrição. Outro fator que exerce um controle extraordinário no comportamento celular, regulando a transcrição gênica e influenciando diversos processos biológicos, e que, quando alterado, é associado ao processo de tumorigênese da mama é a matriz extracelular. A laminina, um dos principais componentes da matriz extracelular, interage com as células através das integrinas e está relacionada ao fenótipo maligno, atuando na adesão, migração, proliferação, diferenciação e sobrevivência celular. Nosso grupo identificou diversos genes diferencialmente expressos em células de câncer de mama ER+ na presença ou ausência de uma monocamada de laminina utilizando a técnica DDRT-PCR. Dois dos genes identificados, DDEF2 e PHLDA1, estão associados à adesão; DDEF2 envolvido na sinalização das integrinas e PHLDA1 relacionado com apoptose por perda de adesão. Nosso objetivo foi investigar os efeitos do 17-estradiol e da laminina na regulação da expressão dos genes DDEF2 e PHLDA1 nas linhagens celulares MCF-7...

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach

Alves-Costa, Fernanda A.; Figueiredo, Márcio A.; Lanes, Carlos F.C.; Almeida, Daniela V.; Marins, Luis F.; Wasko, Adriane Pinto
Fonte: Academia Brasileira de Ciências Publicador: Academia Brasileira de Ciências
Tipo: Artigo de Revista Científica Formato: 487-494
ENG
Relevância na Pesquisa
27.68%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); A presença de níveis mais elevados do hormônio de crescimento (GH) em animais transgênicos poderia levar a várias alterações fisiológicas. Uma linhagem transgênica de paulistinha (Danio rerio) para o GH foi comparada com amostras não transgênicas (NT) desta espécie, através de uma abordagem de DDRT-PCR, com o objetivo de identificar transcritos candidatos diferencialmente expressos em tecido cerebral que poderiam estar envolvidos na superexpressão de GH. Análises densitométricas de dois produtos de amplificação selecionados, p300 e ADCY2, apontaram uma expressão gênica significativamente menor nas amostras transgênicas de paulistinha (104.02 ± 57.71; 224.10 ± 91.73), quando comparadas com as amostras NT (249.75 ± 30.08; 342.95±65.19). Os presentes dados indicam que p300 e ADCY2 estão envolvidos em um sistema de regulação do GH, quando altos níveis circulantes desse hormônio são encontrados em paulistinha.; The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach...

Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells

De Almeida, Danilo C.; Santos, Rodrigo Da S.; Ribeiro-Paes, João T.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 424-431
ENG
Relevância na Pesquisa
38.2%
Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR. Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences in band distribution in SDS-Page gels. Finally through gene expression analysis...

Dental development of children and adolescents with cleft lip and palate

Topolski, Francielle; Souza, Rafael Boscheti de; Franco, Ademir; Cuoghi, Osmar Aparecido; Assunção, Luciana Reichert Da Silva; Fernandes, Ângela
Fonte: Faculdade de Odontologia de Piracicaba - UNICAMP Publicador: Faculdade de Odontologia de Piracicaba - UNICAMP
Tipo: Artigo de Revista Científica Formato: 319-324
ENG
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17.34%
Aim: To evaluate the dental development of Brazilian children and adolescents with cleft lip and palate. Methods: The sample consisted of 107 panoramic radiographs of children and adolescents with cleft lip and/or palate (cleft group) and 107 panoramic radiographs of children and adolescents without cleft lip and/or palate (control group), with chronological ages ranging from 6 to 15 years, matched in gender and chronological age within 60 days. Radiographs were digitized and masked and dental age was assessed using the method described by Demirjian et al. (1973). Three trained examiners conducted the assessments. Each examiner evaluated the radiographs three times. Data were statistically analyzed using non-parametric tests and univariate linear regression (p<0.05). Results: The dental age was overestimated in relation to the chronological age in both groups (p<0.0001). Compared to the control group, there was a delay in the dental age in the cleft group of 0.17 years (2.1 months). However, no statistically significant difference in the dental age between the cleft and the control group was found even when considering the different cleft types (p=0.152). Conclusions: There was no statistically significant difference in the dental age between the cleft and the control groups. The evaluation of dental development in individuals with cleft lip and palate should be approached in the same way as in individuals without clefts...

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues

Alves-costa, F. A.; Wasko, A. P.
Fonte: Instituto Internacional de Ecologia Publicador: Instituto Internacional de Ecologia
Tipo: Artigo de Revista Científica Formato: 224-228
ENG
Relevância na Pesquisa
28.02%
Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.; Display Diferencial (DD) é uma técnica amplamente utilizada em estudos de expressão diferencial. A maioria desses estudos envolvendo espécies de peixes está restrita a espécies da América do Norte e Europa ou a espécies comerciais...

Identificação de genes diferencialmente expressos nas castas da rainha e operaria de Melipona quadrifasciata; Identification of differentially expressed genes in queen and worker castes of Melipona quadrifasciata

Carla Cristina Maria Judice
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 13/04/2005 PT
Relevância na Pesquisa
17.34%
O polifenismo de castas é um fenômeno multifacetado, sendo mais evidente em diferenças na capacidade reprodutiva e na longevidade entre rainhas e operárias. No presente estudo, a abelha sem ferrão Melipona quadrifasciata anthidioides foi escolhida para a investigação de diferenças na expressão gênica entre rainhas e operárias recém nascidas. Este estudo foi focalizado em adultos recém nascidos pois esse estágio representa o ponto final do desenvolvimento embrionário e o ponto inicial para o desempenho de tarefas casta-específicas. Inicialmente, foi empregada a metodologia do ?differential display reverse transcription (DDRT)-PCR? e podendo ser detectada a expressão diferenciada de 14 genes entre rainhas e operárias. As diferenças no perfil de expressão foram posteriormente analisadas através de duas bibliotecas subtrativas de cDNA: uma representando genes muito expressos ou induzidos em operárias quando em comparação com rainhas, e a outra representando genes mais expressos na casta rainha. Embora o número total de seqüências geradas não tenha permitido uma compreensão completa dos transcriptomas das castas, foi possível detectar em rainhas a predominância de genes envolvidos na regulação transcricional e no metabolismo...

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development

Santana,Flávia A.; Nunes,Francis M.F.; Vieira,Carlos U.; Machado,Maria Alice M.S.; Kerr,Warwick E.; Silva Jr,Wilson A.; Bonetti,Ana Maria
Fonte: Academia Brasileira de Ciências Publicador: Academia Brasileira de Ciências
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2006 EN
Relevância na Pesquisa
27.34%
We have compared gene expression, using the Differential Display Reverse Transcriptase - Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach

Alves-Costa,Fernanda A.; Figueiredo,Márcio A.; Lanes,Carlos F.C.; Almeida,Daniela V.; Marins,Luis F.; Wasko,Adriane P.
Fonte: Academia Brasileira de Ciências Publicador: Academia Brasileira de Ciências
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2012 EN
Relevância na Pesquisa
27.34%
The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.

Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

Silva,A.M.; Pires,E.G.; Abrantes,E.F.; Ferreira,L.R.P.; Gazzinelli,R.T.; Reis,L.F.L.
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 EN
Relevância na Pesquisa
27.88%
The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion...

Evaluation of Parkia pendula lectin mRNA differentially expressed in seedlings

Rêgo,MJBM.; Santos,PB.; Carvalho-Junior,LB.; Stirling,J.; Beltrão,EIC.
Fonte: Instituto Internacional de Ecologia Publicador: Instituto Internacional de Ecologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/05/2014 EN
Relevância na Pesquisa
27.34%
Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class.

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues

Alves-Costa,FA.; Wasko,AP.
Fonte: Instituto Internacional de Ecologia Publicador: Instituto Internacional de Ecologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2015 EN
Relevância na Pesquisa
27.68%
Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology

Sturtevant, Joy
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2000 EN
Relevância na Pesquisa
18.02%
The host-fungus interaction is characterized by changes in gene expression in both host and pathogen. Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis of gene expression among several cell populations. Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression. Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported. DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets. In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes. In Candida albicans, DDRT-PCR studies identified TIF-2, which may play a role in the upregulation of phospholipases, and the stress-related genes, CIP1 and CIP2. In Histoplasma capsulatum and C. albicans, genes involved in the host-pathogen interaction, including a member of the 100-kDa family in Histoplasma and an ALS and 14-3-3 gene in Candida...

Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR).

Bauer, D; Müller, H; Reich, J; Riedel, H; Ahrenkiel, V; Warthoe, P; Strauss, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/09/1993 EN
Relevância na Pesquisa
27.34%
We have significantly improved a method originally developed by Liang and Pardee [Science 257 (1992) 967-971] to display a broad spectrum of expressed genes and to detect differences in expression between different cell types. We have analysed various aspects of the technique and have modified it for both, the application to fast and efficient identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also introduced nondenaturating gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of its application.

Gene Expression Profiling of Oral Squamous Cell Carcinoma by Differential Display RT-PCR and Identification of Tumor Biomarkers

Chakraborty, Sanjukta; Nagashri, M. N.; Mohiyuddin, S. M. Azeem; Gopinath, K. S.; Kumar, Arun
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
17.68%
Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Despite progress in therapeutic and surgical treatments, its survival period at 5 years is the lowest among major cancers, and remains unchanged in the last two decades. The growing epidemiological relevance of oral cancer emphasizes the need to better understand the molecular mechanisms underlying this disease and identify predictive tumor markers and therapeutic targets. To this end, we have used the DDRT-PCR analysis to profile the oral tumor transcriptome and identify differentially regulated genes that may be used as potential biomarkers and therapeutic targets. Our DDRT-PCR analysis identified 51 differentially expressed fragments, of which 25 were revalidated by reverse Northern analysis. Northern blot analysis further corroborated these findings for a few genes. In order to ascertain the utility of some of the identified genes as molecular markers and therapeutic targets, semi-quantitative RT-PCR analysis was carried out in a panel of matched oral normal and tumor samples, that confirmed GLTP, PCNA, RBM28, C17orf75 and DIAPH1 as significantly upregulated, whereas TNKS2, PAM and TUBB2C showed significant downregulation in tumor samples. Taken together...

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach

Alves-Costa, Fernanda Antunes; Figueiredo, M??rcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes; Wasko, Adriane Pinto
Fonte: Universidade Federal do Rio Grande Publicador: Universidade Federal do Rio Grande
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
27.68%
The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ?? 57.71; 224.10 ?? 91.73) when compared to NT samples (249.75 ?? 30.08; 342.95 ?? 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.; A presen??a de n??veis mais elevados do horm??nio de crescimento (GH) em animais transg??nicos poderia levar a v??rias altera????es fisiol??gicas. Uma linhagem transg??nica de paulistinha (Danio rerio) para o GH foi comparada com amostras n??o transg??nicas (NT) desta esp??cie, atrav??s de uma abordagem de DDRT-PCR, com o objetivo de identificar transcritos candidatos diferencialmente expressos em tecido cerebral que poderiam estar envolvidos na superexpress??o de GH. An??lises densitom??tricas de dois produtos de amplifica????o selecionados...

Detecção de genes diferencialmente expressos na formiga urbana Camponotus vittatus (Hymenoptera, Formicinae)

Rodovalho, Cynara de Melo
Fonte: Universidade Federal de Uberlândia Publicador: Universidade Federal de Uberlândia
Tipo: Dissertação
POR
Relevância na Pesquisa
27.88%
Insetos sociais despertam grande curiosidade, interesse e uma vontade de se compreender as bases moleculares da vida social. A identificação de genes diferencialmente expressos tem sido usada para o entendimento não só da função gênica, mas também dos mecanismos moleculares de diferentes processos biológicos. Nesse estudo, foram testados dois protocolos para extração de RNA total de Camponotus vittatus e a técnica de Differential Display Reverse Transcriptase – Polymerase Chain Reaction (DDRT-PCR) foi utilizada para a identificação de genes diferencialmente expressos em casta, sexo e nos estágios de desenvolvimento dessa formiga. O método de extração de RNA baseado em Chomczynski e Sacchi (1987) demonstrou ser o mais eficaz. A técnica DDRTPCR foi eficiente para a detecção de genes diferencialmente expressos de C. vittatus. Três expressed sequence tags (ESTs) foram seqüenciadas e análises por bioinformática não detectaram similaridade com seqüências depositadas em banco de dados, podendo ser novas e, talvez, específicas de C. vittatus. Essas seqüências foram depositadas no banco de dados dbEST. Um fragmento diferencialmente expresso em operária foi obtido com a combinação dos primers: HT11G e AP03. Os resultados do seqüenciamento e das análises por bioinformática revelaram que tal EST foi isolada da bactéria endossimbionte de C. vittatus...

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

Venkatesh, B.; Hettwer, U.; Koopmann, B.; Karlovsky, P.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 01/11/2014
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Background: Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols are cDNA Differential Display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. Results: We developed procedures for the conversion of DDRT-PCR data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. Conclusion: We developed a data processing procedure for quantitative analysis of amplified cDNA fragments. The system utilizes common software and provides an open-end alternative to microarray analysis. The processing is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful in research on organisms for which microarray analysis is not available or economical.

Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina; Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells

Almeida, Danilo C. de; Santos, Rodrigo da S.; Ribeiro-Paes, João T.
Fonte: Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto Publicador: Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/publishedVersion; ; Formato: application/pdf
Publicado em 30/12/2012 ENG
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Model of study: Experimental study. Introduction: Recently, stem cell research has generated greatinterest due to its applicability in regenerative medicine. Bone marrow is considered the most importantsource of adult stem cells and the establishment of new methods towards gene expression analysisregarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the geneexpression of different stem cells in distinct situations.Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in globalgene expression of mice bone marrow cells under two conditions.Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week withoutmedium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to geneexpression analyses by DDRT-PCR.Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences inband distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands(1300...