RESUMEN
The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.
Asunto(s)
Proteínas Recombinantes/metabolismo , Proteína S6 Ribosómica/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnica del Anticuerpo Fluorescente , Redes Reguladoras de Genes , Genoma , Genómica , Humanos , Análisis por Micromatrices , Terapia Molecular Dirigida , Fosforilación , Interferencia de ARN , Proteínas Recombinantes/genética , Proteína S6 Ribosómica/genética , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
We have evaluated the performance characteristics of three quantitative gene expression technologies and correlated their expression measurements to those of five commercial microarray platforms, based on the MicroArray Quality Control (MAQC) data set. The limit of detection, assay range, precision, accuracy and fold-change correlations were assessed for 997 TaqMan Gene Expression Assays, 205 Standardized RT (Sta)RT-PCR assays and 244 QuantiGene assays. TaqMan is a registered trademark of Roche Molecular Systems, Inc. We observed high correlation between quantitative gene expression values and microarray platform results and found few discordant measurements among all platforms. The main cause of variability was differences in probe sequence and thus target location. A second source of variability was the limited and variable sensitivity of the different microarray platforms for detecting weakly expressed genes, which affected interplatform and intersite reproducibility of differentially expressed genes. From this analysis, we conclude that the MAQC microarray data set has been validated by alternative quantitative gene expression platforms thus supporting the use of microarray platforms for the quantitative characterization of gene expression.
Asunto(s)
Perfilación de la Expresión Génica/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Garantía de la Calidad de Atención de Salud/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.
Asunto(s)
Análisis de Falla de Equipo/métodos , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , ARN/análisis , ARN/genética , Algoritmos , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estados UnidosRESUMEN
BACKGROUND: Image-based screens can produce hundreds of measured features for each of hundreds of millions of individual cells in a single experiment. RESULTS: Here, we describe CellProfiler Analyst, open-source software for the interactive exploration and analysis of multidimensional data, particularly data from high-throughput, image-based experiments. CONCLUSION: The system enables interactive data exploration for image-based screens and automated scoring of complex phenotypes that require combinations of multiple measured features per cell.