Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
Mol Biotechnol ; 34(3): 303-15, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17284778

RESUMO

As the quality of microarrays is critical to successful experiments for data consistency and validity, a reliable and convenient quality control method is needed. We describe a systematic quality control method for large-scale genome oligonucleotide arrays. This method is comprised of three steps to assess the quality of printed arrays. The first step involves assessment of the autofluorescence property of DNA. This step is convenient, quick to perform, and allowed reuse of every array. The second step involves hybridization of arrays with Cy3-labeled 9-mer oligonucleotide target to assess the quality and stability of oligonucleotides. Because this step consumed arrays, one or two arrays from each batch were used to complement the quality control data from autofluorescence. The third step involves hybridization of arrays from every batch with transcripts derived from two cell lines to assess data consistency. These hybridizations were able to distinguish two closely related tissue samples by identifying a cluster of 20 genes that were differently expressed in U87MG and T98G glioblastoma cell lines. In addition, we standardized two parameters that significantly enhanced the quality of arrays. We found that longer pin contact time and crosslinking oligonucleotides at 400 mJ/cm(2) were optimal for the highest hybridization intensity. Taken together, these results indicate that the quality of spotted oligonucleotide arrays should be assessed by at least two methods, autofluorescence and 9-mer hybridization before arrays are used for hybridization experiments.


Assuntos
Perfilação da Expressão Gênica/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/normas , Animais , Encéfalo/patologia , Encéfalo/virologia , Química Encefálica , Carbocianinas/análise , Linhagem Celular Tumoral/química , DNA Complementar/genética , DNA de Neoplasias/genética , Encefalomielite Equina Venezuelana/patologia , Fluorescência , Corantes Fluorescentes/análise , Fluorometria , Glioblastoma/patologia , Humanos , Camundongos , Proteínas de Neoplasias/genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Sondas de Oligonucleotídeos/efeitos da radiação , Polilisina , Controle de Qualidade , Técnica de Subtração , Fatores de Tempo , Transcrição Gênica , Raios Ultravioleta
2.
BMC Dev Biol ; 5: 22, 2005 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-16207381

RESUMO

BACKGROUND: The identification of molecular pathways of differentiation of embryonic stem cells (hESC) is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. RESULTS: Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than > or = 3 folds in human embryoid bodies (hEB). These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS) analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs) were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. CONCLUSION: These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.


Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Perfilação da Expressão Gênica/métodos , Células-Tronco/citologia , Biomarcadores , Análise por Conglomerados , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
3.
Stem Cells Dev ; 14(3): 270-84, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15969622

RESUMO

Human embryonic stem cells (hESC) must be differentiated before clinical use. In addition, the extent of contamination of undifferentiated cells and the efficiency of differentiation must also be assessed prior to clinical application. In this manuscript, we describe the development of a focused microarray that may be used to discriminate between hESC and their differentiated progeny. This array contains 755 genes including embryonic stem cell markers as well as markers of differentiation into neural, mesodermal, and endodermal phenotypes. In addition, we have included candidate genes belonging to families of cytokines, chemokines, receptors, signaling pathways, and homeodomain proteins that are likely to be important in the process of differentiation. Testing and validation of the focused array was performed using RNA from hESC, human embryoid body (hEB) outgrowths, and a human embryonal carcinoma (hEC) cell line. We have compared gene expression with negative background, GAPDH, beta-actin positive controls, and human universal RNA (hURNA), showing that such an array can rapidly distinguish between undifferentiated and differentiated hESC-derived cell populations. We expect that the described array will be extremely useful in evaluating the extent of differentiation and the state of the hESC-derived population utilized for therapeutic purposes.


Assuntos
Diferenciação Celular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células-Tronco/citologia , Linhagem Celular , Embrião de Mamíferos , Enzimas/genética , Humanos , Hibridização de Ácido Nucleico , Proteínas/genética , RNA/genética , RNA/isolamento & purificação , Transplante de Células-Tronco , Células-Tronco/fisiologia
4.
J Immunother ; 35(1): 23-31, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22130162

RESUMO

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, ß, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.


Assuntos
Adenocarcinoma/imunologia , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neoplasias Ovarianas/imunologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Interferon-alfa/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/genética , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transgenes/genética
5.
J Immunother ; 32(8): 803-16, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19752753

RESUMO

A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-alpha. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. To enhance the antiproliferative activity of IFN-alpha, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-alpha and IFN-gamma, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-alpha among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-alpha, whereas it partially inhibited that of IFN-gamma. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through TRAIL-receptor (R) 1 and TRAIL-R2. In IFN-alpha-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL whereas Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-alpha predominantly required IRF9. Furthermore, IFN-stimulated response element-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex after IFN-alpha treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-alpha and TRAIL, suggesting that TRAIL-R2 mediated both IFN-alpha and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-alpha-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-alpha. Thus, this study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-alpha and TRAIL may be one of the potential mediators.


Assuntos
Adenocarcinoma/imunologia , Glioblastoma/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Interferon-alfa/farmacologia , Neoplasias Ovarianas/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Farmacológicos , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Fator Gênico 3 Estimulado por Interferon/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Interferon gama/farmacologia , Janus Quinases/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
6.
Stem Cells ; 22(3): 292-312, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15153607

RESUMO

Human ES (hES) cell lines have only recently been generated, and differences between human and mouse ES cells have been identified. In this manuscript we describe the properties of two human ES cell lines, BG01 and BG02. By immunocytochemistry and reverse transcription polymerase chain reaction, undifferentiated cells expressed markers that are characteristic of ES cells, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4. Both cell lines were readily maintained in an undifferentiated state and could differentiate into cells of all three germ layers, as determined by expression of beta-tubulin III neuron-specific molecule (ectoderm), cardiac troponin I (cardiomyocytes, mesoderm), and alpha-fetoprotein (endoderm). A large-scale microarray (16,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG01 and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE01, GE09, and pooled samples derived from GE01, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming growth factor-beta superfamily were employed to examine differentially expressed genes. Several growth factors, receptors, and components of signaling pathways that regulate embryonic development, in particular the nodal signaling pathway, were detected in both BG01 and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG01 and BG02 human ES cell lines.


Assuntos
Diferenciação Celular , Linhagem Celular , Células-Tronco Pluripotentes , Transdução de Sinais , Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Receptores de Fatores de Crescimento/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo
7.
Blood ; 103(8): 2956-64, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15070671

RESUMO

Human embryonic stem (huES) cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation of huES cells and factors regulating cell development. We have used high-quality microarrays containing 16 659 seventy-base pair oligonucleotides to examine gene expression in 6 of the 11 available huES cell lines. Expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All 6 cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient > 0.85).A common subset of 92 genes was identified that included Nanog, GTCM-1, connexin 43 (GJA1), oct-4, and TDGF1 (cripto). Gene expression was confirmed by a variety of techniques including comparison with databases, reverse transcriptase-polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry. Comparison with published "stemness" genes revealed a limited overlap, suggesting little similarity with other stem cell populations. Several novel ES cell-specific expressed sequence tags were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES cells and provide a unique set of markers to profile and better understand the biology of huES cells.


Assuntos
Expressão Gênica , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Biologia Computacional , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA