RESUMEN
Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa/métodos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Hígado/enzimología , Miocardio/enzimología , Fosfotransferasas/biosíntesis , Fosfotransferasas/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Target validation in health and disease integrates the modulation of a certain molecular target with an expected biological/biochemical/physiological or pathophysiological response or effect. The current state-of-the-art in target validation requires the interface of multiple complementary approaches and technologies to define the mechanistic connectivity between a molecular target and underlying micro- and macrobiotic processes. Target validation also represents the basis for "drug target validation" with focus on therapeutic applications. The concepts of "target validation" and "drug-based therapeutic intervention" continue to coevolve as new classes of therapeutic agents and delivery systems emerge and enable us to target or modulate previously inaccessible molecular entities.
Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Proteómica , Animales , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Proteómica/instrumentación , Proteómica/métodos , Tecnología Farmacéutica/instrumentación , Tecnología Farmacéutica/métodosRESUMEN
Synucleins are proteins known for their malfunction in a group of illnesses called synucleopathies, which includes Alzheimer's and Parkinson's disease. To learn more about the role of synucleins in the CNS, we have studied levels of message coding for alpha-, beta-, and gamma-synuclein using quantitative RT-PCR. Levels of synuclein mRNAs were studied in the cerebral cortex (left and right, anterior and posterior), hippocampus, striatum, and cerebellum, obtained from 5-d-old (newborn), 1-mo (juvenile)-, and 6-, and 9-mo (adult)-old rats. The mRNA levels for all synucleins varied significantly among structures. The rank order of mRNA levels in different structures was cortex = hippocampus > striatum > cerebellum for alpha-synuclein; cortex > hippocampus = cerebellum > striatum for beta-synuclein; and hippocampus = striatum > cortex = cerebellum for gamma-synuclein. There was significant effect of age for mRNA levels for all synucleins. The dynamics of these changes were different depending on type of synuclein and brain structure. Levels of mRNA for alpha-synuclein were significantly reduced with age in all structures except hippocampus. For beta- and gamma-synuclein, levels increased significantly only in the cerebral cortex and only from 5 d to 1 mo of age. In contrast, gamma-synuclein levels in the cerebellum were very high at 5 d and significantly reduced at 1 mo of age. The revealed pattern and dynamics of changes in the levels of mRNA coding for synucleins would support the conclusion for an important role of these molecules during development and the aging process.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , ARN Mensajero/biosíntesis , Ratas/metabolismo , alfa-Sinucleína/genética , Sinucleína beta/genética , gamma-Sinucleína/genética , Envejecimiento/genética , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Cuerpo Estriado/crecimiento & desarrollo , Cuerpo Estriado/metabolismo , Femenino , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Masculino , Especificidad de Órganos , Ratas/genética , Ratas/crecimiento & desarrollo , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-Sinucleína/biosíntesis , Sinucleína beta/biosíntesis , gamma-Sinucleína/biosíntesisRESUMEN
The emerging field of biomarkers has applications in the diagnosis, staging, prognosis and monitoring of disease progression, as well as in the monitoring of clinical responses to a therapeutic intervention and the development and delivery of personalized treatments to reduce attrition in clinical trials. Moreover, biomarkers have a positive impact on health economics. The word "biomarker" has been used extensively across therapeutic areas and many disciplines, and its nature takes into consideration clinical, physiological, biochemical, developmental, morphological and molecular measures. In drug trials, biomarkers have been proposed for use in efficacy determination and patient population stratification, in deducing pharmacokinetic-pharmacodynamic relationships and in safety monitoring. The interfacing and integration of different technologies for data collection and analysis are pivotal to biomarker identification, characterization, validation and application. "Integrative functional informatics" represents a novel direction in such technology integration.
Asunto(s)
Biomarcadores , Biología Computacional , Recolección de Datos , Genómica , Proteómica , HumanosRESUMEN
The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , ARN Interferente Pequeño/genética , Secuencia de Bases , Línea Celular , Biología Computacional , Evaluación Preclínica de Medicamentos/instrumentación , Genómica , Humanos , Reacción en Cadena de la Polimerasa/métodos , Robótica , TransfecciónRESUMEN
Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.
Asunto(s)
Adenoviridae , Vectores Genéticos , Proteínas Recombinantes/análisis , Biología ComputacionalRESUMEN
The biopharmaceutical industry is currently being presented with opportunities to improve research and business efficiency via automation and the integration of various systems. In the examples discussed, industrial high-throughput screening systems are integrated with functional tools and bioinformatics to facilitate target and biomarker identification and validation. These integrative functional approaches generate value-added opportunities by leveraging available automation and information technologies into new applications that are broadly applicable to different types of projects, and by improving the overall research and development and business efficiency via the integration of various systems.
Asunto(s)
Automatización , Biología Computacional , Informática , Genómica/métodos , Genómica/tendencias , Humanos , Tecnología Farmacéutica/métodosRESUMEN
To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Recuento de Células , Células Cultivadas , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Células K562 , Poli A/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificaciónRESUMEN
To meet growing needs for high throughput gene expression profiling, we established a new automated high throughput TaqMan RT-PCR method for quantitative mRNA expression analysis. In this method, the Allegro( trade mark ) (Zymark) system conducts all sample tracking and liquid handling steps, and ABI PRISM 7900 HT (Applied Biosystems) is used to conduct real-time determination of the C(t) value when amplification of PCR products is first detected and accumulation of inhibitory PCR products is unlikely to occur. The ABI PRISM 7900 HT Sequence Detection System features a real-time PCR instrument with 384-well-plate compatibility and robotic loading, and continuous wavelength detection, which enables the use of multiple fluorophores in a single reaction. The Allegro System offers an assembly line approach with a modular design that allows reconfiguration of the components to accommodate variations in the assay flow. In the present study, we have established and validated a new automated High Throughput (HT) TaqMan RT-PCR- based method for quantitative mRNA expression analysis. The data demonstrate that HT-Taqman PCR is a powerful tool that can be used for measuring low concentrations of mRNA, and is highly accurate, reproducible, and amenable to high throughput analysis. Results suggest that HT-TaqMan is a reliable method for the quantification of low-expression genes and a powerful tool with HT capability for target identification/validation, structure-activity relationship (SAR) study, compound selection for efficacy studies, and biomarker identification in drug discovery and development.
Asunto(s)
Perfilación de la Expresión Génica/métodos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Diseño de Fármacos , Perfilación de la Expresión Génica/instrumentación , ARN Mensajero/biosíntesis , Polimerasa Taq/metabolismoRESUMEN
Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the intrinsic analytical precision, large dynamic range, and high sensitivity of RT-qPCR, yet is scalable to high throughputs. We have evaluated the performance of a nanoliter fluidic system that enables up to 3072 nanoliter RT-qPCR assays simultaneously in a high-density array format. We measured the transcription from two different adult human tissues to assess measurement reproducibility across replicates, measurement accuracy, precision, specificity, and sensitivity; determined the false positive rate (FPR) and false negative rate (FNR) of the expressed transcript copies; and determined differences in kinase gene expression reflecting tissue and dosage differences. Using our methodology, we confirm the potential of this technology in advancing pharmaceutical research and development.
Asunto(s)
Nanotecnología/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacciones Falso Negativas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hígado/metabolismo , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente PrincipalRESUMEN
Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Perfilación de la Expresión Génica/métodos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Industria Farmacéutica/métodos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Análisis por Matrices de Proteínas/métodos , Proteína Quinasa C/análisis , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los ResultadosRESUMEN
PURPOSE OF REVIEW: The onset of cancer anorexia and the accompanying neurological symptoms and signs involve the general influence of cytokines on the brain. Using methylcholanthrene to induce tumors in Fischer 344 rats, we measured various specific components of the cytokine-induced anorectic reaction, including: (1) IL-1beta system components (ligand, signaling receptor, receptor accessory proteins, and receptor antagonist); (2) TNF-alpha; (3) TGF-beta1; and (4) IFN-gamma in the tumor tissue, the liver and the brain. RECENT FINDINGS: The data show that IL-1beta, TNF-alpha and IFN-gamma messenger RNA were detected in the tumor tissue of anorectic tumor-bearing rats. In brain regions, anorexia is associated with the upregulation of IL-1beta and its receptor mRNA. All other mRNA remained unchanged in the brain regions examined. SUMMARY: This suggests that IL-1beta and its receptor may play a significant role in this model of cancer-associated anorexia. In vivo, the characterization of cytokine components in the brain may provide data for potential pharmacological interventions to ameliorate the anorexia of disease.