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1.
Anal Biochem ; 352(1): 50-60, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16545767

RESUMO

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.


Assuntos
Ensaio de Amplificação de Sinal de DNA Ramificado/métodos , Perfilação da Expressão Gênica/métodos , Processamento Alternativo , Apoptose , Corantes Fluorescentes , Regulação da Expressão Gênica , Células HeLa , Humanos , Inflamação/metabolismo , Modelos Biológicos , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Células U937
2.
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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