Assuntos
Bacteriófago T4/enzimologia , Sondas de DNA/análise , Nylons , Análise de Sequência com Séries de Oligonucleotídeos , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Trifosfato de Adenosina/metabolismo , Bacteriófago T4/genética , Soluções Tampão , Reagentes de Ligações Cruzadas/metabolismo , Sondas de DNA/metabolismo , Etiquetas de Sequências Expressas/química , Etiquetas de Sequências Expressas/metabolismo , Genes Bacterianos , Isótopos de Fósforo , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo 5'-Hidroxiquinase/isolamento & purificação , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Raios UltravioletaRESUMO
To date, most studies of multigenic expression patterns by long DNA array have used DNA fragments as probes. These probes are usually obtained as PCR products, and this represents a time-consuming and error-prone approach, requiring strict quality control. The present study examines the use of 40- and 70-mer synthetic oligonucleotides as probes for DNA array analysis with radioactive labeled targets. Design, spotting onto nylon filters, and hybridization conditions were determined and optimized. In this approach, the sensitivity and the specificity of the hybridization appear comparable to the conventional long DNA probes assay, permitting the analysis of small samples of approximately 1 microg total RNA. The long oligonucleotide array thus provides a very convenient method for the analysis of gene expression patterns in biological specimens and in clinical research.
Assuntos
Sondas de DNA , Perfilação da Expressão Gênica/instrumentação , Teste de Materiais , Nylons , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Arabidopsis/genética , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Humanos , Marcação por Isótopo/métodos , Membranas Artificiais , Isótopos de Fósforo , Controle de Qualidade , RNA/análise , RNA/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Several studies have supported the hypothesis that adrenocortical tumor formation is the result of a multistep process. The angiogenic switch has been proposed to be a key step in tumor progression from adenoma to carcinoma. In this study we measured the cytosolic concentrations of three proteins involved in angiogenesis [namely platelet-derived endothelial cell growth factor vascular endothelial cell growth factor A (VEGF-A), and thrombospondin-1 (TSP1)] in a series of 43 human sporadic adrenocortical tumors. The tumors were classified as adenomas (n = 18), transitional tumors (n = 12), or carcinomas (n = 13) according to the histological criteria defined by Weiss. Platelet-derived endothelial cell growth factor/thymidine phosphorylase levels were not significantly different among these three groups. One hundred percent of the adenomas and 73% of the transitional tumors showed VEGF-A concentrations under the threshold value of 107 ng/g protein, whereas 75% of the carcinomas had VEGF-A concentrations above this threshold value. Similarly, 89% of the adenomas showed TSP1 concentrations above the threshold value of 57 microg/g protein, whereas only 25% of the carcinomas and 33% of the transitional tumor samples did so. Insulin-like growth factor II overexpression, a common genetic alteration of adrenocortical carcinomas, was significantly correlated with higher VEGF-A and lower TSP1 concentrations. The tumors from the 6 patients with tumor recurrence after surgical ablation showed significantly higher VEGF-A values than the carcinomas and the transitional tumors from patients that did not relapse. Taken together, these data suggest that a decrease in TSP1 expression is an event that precedes an increase in VEGF-A expression during adrenocortical tumor progression. The population of premalignant tumors with low TSP1 and normal VEGF-A levels could represent a selective target for antiangiogenic therapies.
