RESUMO
Large-scale microarray expression profiling studies have helped us to understand basic biological processes and to classify and predict the prognosis of cancers; they have also accelerated the identification of new drug targets. Affymetrix GeneChip probe arrays are high-density oligonucleotide microarrays that are available for many prokaryotic and eukaryotic species. Affymetrix human and mouse whole-genome microarrays analyze the expression level of up to 47,000 transcripts and variants. Each transcript is measured by 11 probe pairs, which consist of a perfect match 25mer oligonucleotide (PM) and a 25mer mismatch oligonucleotide (MM) that contains a single base pair mismatch in the central position. The PM/MM design is used for identification and subtraction of nonspecific hybridization and background signals. Advantages of Affymetrix GeneChip arrays include highly standardized array fabrication, as well as standardized target preparation, hybridization, and processing protocols, resulting in low technical variability and good reproducibility between experiments. This chapter describes the standard assay procedures for isolating total RNA from heart tissue, generating a biotin-labeled target for expression analysis, processing of Affymetrix GeneChip probe arrays using the Affymetrix instrument system, and quality control measures.
Assuntos
Perfilação da Expressão Gênica/métodos , Coração/fisiologia , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Animais , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , RatosRESUMO
Heart failure is characterized at the cellular level by impaired contractility and abnormal Ca2+ homeostasis. We have previously shown that restoration of a key enzyme that controls intracellular Ca(2+) handling, the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), induces functional improvement in heart failure. We used high-density oligonucleotide arrays to explore the effects of gene transfer of SERCA2a on genetic reprogramming in a model of heart failure. A total of 1,300 transcripts were identified to be unmodified by the effect of virus alone. Of those, 251 transcripts were found to be up- or down-regulated upon failure. A total of 51 transcripts which were either up--(27) or down--(24) regulated in heart failure were normalized to the nonfailing levels by the restoration of SERCA2a by gene transfer. The microarray analysis identified new genes following SERCA2a restoration in heart failure, which will give us insights into their role in the normalization of multiple pathways within the failing cell.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Baixo Débito Cardíaco/genética , Baixo Débito Cardíaco/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Miocárdio/metabolismo , Transcrição Gênica/genética , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , Regulação para Baixo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Homeostase , Humanos , Ratos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Regulação para CimaRESUMO
A frequent characteristic of human papillomavirus (HPV)-positive cervical cancers is the loss of viral E2 gene expression in HPV-infected cervical epithelial cells as a consequence of viral DNA integration into the cellular genome. The expression of E2 in HPV-positive cancer cells results in the repression of the viral E6/E7 oncogenes, activation of the p53 and pRB pathways, and a G1 cell cycle arrest, followed by induction of cellular senescence. The transcriptional consequences of E2-mediated cell cycle arrest that lead to senescence currently are unknown. Using conditional senescence induction in HeLa cells and microarray analysis, we describe here the expression profile of cells irreversibly committed to senescence. Our results provide insight into the molecular anatomy of senescence pathways and its regulation by HPV on-coproteins. These include the induction of the RAB vesicular transport machinery and a general down-regulation of chromatin regulatory molecules. The repression of tumor-specific G antigens during E2 senescence supports a reversal of the tumorigenic phenotype by E2 and the potential approach of tumor-specific G antigen-specific immunotherapy for cervical cancer.