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1.
Methods Mol Biol ; 356: 253-65, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16988409

RESUMO

The use of photoremovable protecting groups in biology affords the end user high temporal, spatial, and concentration control of reagents and substrates. High content screening and other large-scale biology applications would benefit greatly from these advantages. Herein, we report progress in this field by highlighting the recent development of controllable siRNA (csiRNA), which is a dormant siRNA that can be activated using 365 nm light. Two different experimental designs are described to highlight the temporal and concentration variables that can be controlled. First, the RNAi process is activated at two timepoints, 24- and 48-h post-transfection, to demonstrate that the action of csiRNA does not begin until activated. Second, increasing light dosage exposure to cells transfected with csiRNA that controls the concentration of active siRNA molecules. All experiments are conducted in a 96-well format with light delivered through the UCOM device.


Assuntos
Análise Serial de Tecidos/métodos , Análise Serial de Tecidos/tendências , Actinas/metabolismo , Relação Dose-Resposta à Radiação , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/deficiência , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Células HeLa , Humanos , Indicadores e Reagentes , Luz , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/efeitos da radiação , Fatores de Tempo , Análise Serial de Tecidos/instrumentação , Transfecção
2.
Biochim Biophys Acta ; 1758(3): 394-403, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16497269

RESUMO

Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.


Assuntos
Expressão Gênica/efeitos dos fármacos , Luz , RNA Interferente Pequeno/química , RNA Interferente Pequeno/efeitos da radiação , Transfecção , Bioensaio , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/administração & dosagem , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/genética , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fenótipo , RNA Interferente Pequeno/administração & dosagem , Survivina , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética
3.
J Biomol Screen ; 10(6): 549-56, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16103413

RESUMO

The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1alpha (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes' expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.


Assuntos
DNA/análise , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Interferente Pequeno/metabolismo , RNA/análise , Citocinas/metabolismo , Primers do DNA/química , Expressão Gênica , Técnicas Genéticas , Células HeLa , Humanos , Interleucina-8/metabolismo , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Acetato de Tetradecanoilforbol/farmacologia , Células U937
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