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1.
G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.  相似文献   

2.
MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression.  相似文献   

3.
Haab BB  Dunham MJ  Brown PO 《Genome biology》2001,2(2):research0004.1-research000413

Background  

We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes.  相似文献   

4.

Background

We describe a method for printing protein microarrays, and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, and the other representing an experimental sample in which the concentrations of specific proteins were to be measured, were labeled by covalent attachment of spectrally-resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signals representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To characterize the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs.

Results

50% of the arrayed antigens, and 20% of the arrayed antibodies, provided specific and accurate measurements of their cognate ligands at or below concentrations of 1.6 µg/ml and 0.34 µg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of less than 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.

Conclusions

Protein microarrays can provide a simple and practical means to characterize patterns of variation in hundreds or thousands of different proteins, in clinical or research applications.  相似文献   

5.
Second-generation sequencing now provides the potential for low-cost generation of whole-genome sequences. However, for large-genome organisms with high repetitive DNA content, genome-wide short read sequence assembly is currently impossible, with accurate ordering and localization of genes still relying heavily on integration with physical and genetic maps. To facilitate this process, we have used Agilent microarrays to simultaneously address thousands of gene sequences to individual BAC clones and contiguous sequences that form part of an emerging physical map of the large and currently unsequenced 5.3-Gb barley genome. The approach represents a cost-effective, highly parallel alternative to traditional addressing methods. By coupling the gene-to-BAC address data with gene-based molecular markers, thousands of BACs can be anchored directly to the genetic map, thereby generating a framework for orientating and ordering genes, and providing direct links to phenotypic traits.  相似文献   

6.
7.
Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.  相似文献   

8.
9.
'Reverse-phase' protein lysate microarray (RPA) assays use micro-scale, cell lysate dot blots that are printed to a substrate, followed by quantitative immunochemical protein detection, known to be particularly effective across many samples. Large-scale sample collection is a labor-intensive and time-consuming process; the information yielded from RPA assays, however, provides unique opportunities to experimentally interpret theoretical protein networks quantitatively. When specific antibodies are used, RPA can generate 1,000 times more data points using 10,000 times less sample volume than an ordinary western blot, enabling researchers to monitor quantitative proteomic responses for various time-scale and input-dose gradients simultaneously. Hence, the RPA system can be an excellent method for experimental validation of theoretical protein network models. Besides the initial screening of primary antibodies, collection of several hundreds of sample lysates from 1- to 8-h periods can be completed in approximately 10 d; subsequent RPA printing and signal detection steps require an additional 2-3 d.  相似文献   

10.
Molecular and functional analysis using live cell microarrays   总被引:1,自引:0,他引:1  
Understanding cellular behavior in both healthy and diseased states requires the ability to molecularly delineate the characteristics of individual cells from complex mixtures. The recent development of cellular microarrays allows such an undertaking. By immobilizing different cell capture and analysis reagents on a solid support, mixtures of cells can be rapidly interrogated for their composition and phenotype. Thus, one can identify and quantitate distinct cell types based on the expression of particular cell surface molecules, as well as analyze their response to defined signals through the secretion of specific factors or other measurable cellular activities. This review focuses on the use of cellular microarrays to detect antigen-specific T cells and their responsiveness, analyze cancer cell types and behavior and to investigate the control of stem cell differentiation.  相似文献   

11.
Na K  Lee M  Shin B  Je Y  Hyun J 《Biotechnology progress》2006,22(1):285-287
To infect cells with a particular multiplicity of infection, it is essential to know the concentration of virus in the inoculum. Here we describe a highly reliable and controllable method for plaque purification using cell-repellent surfaces micropatterned on the substrate. Micropatterning of localized chemical or biochemical domains has the potential to become a powerful tool in controlling the seeding of cells. The cell array was reliably fabricated with micropatterned surfaces, and the number of cells in a pattern was easily controlled by the cell density in the media and micropattern size. The cell micropatterns were infected with baculoviruses to form an array of virus plaques. GFP-modified and wild-type baculoviruses were used to verify the feasibility of purifying a specific plaque. Using confocal microscopy, GFP-expressing plaques were readily selectable and removable.  相似文献   

12.
There are almost 1,300 entries for higher eukaryotes in the Nuclear Protein Database. The proteins' subcellular distribution patterns within interphase nuclei can be complex, ranging from diffuse to punctate or microspeckled, yet they all work together in a coordinated and controlled manner within the three-dimensional confines of the nuclear volume. In this review we describe recent advances in the use of quantitative methods to understand nuclear spatial organisation and discuss some of the practical applications resulting from this work.  相似文献   

13.
MOTIVATION: While the use of cDNA microarrays for functional genomic analysis has become commonplace, relatively little attention has been placed on false positives, i.e. the likelihood that a change in measured radioactive or fluorescence intensity may reflect a change in gene expression when, in fact, there is none. Since cDNA arrays are being increasingly used to rapidly distinguish biomarkers for disease detection and subsequent assay development (Wellman et al., Blood, 96, 398-404, 2000), the impact of false positives can be significant. For the use of this technology, it is necessary to develop quantitative criteria for reduction of false positives with radioactively-labeled cDNA arrays. RESULTS: We used a single source of RNA (HuT78 T lymphoma cells) to eliminate sample variation and quantitatively examined intensity ratios using radioactively labeled cDNA microarrays. Variation in intensity ratios was reduced by processing microarrays in side-by-side (parallel mode) rather than by using the same microarray for two hybridizations (sequential mode). Based on statistical independence, calculation of the expected number of false positives as a function of threshold showed that a detection limit of [log(2)R] >0.65 with agreement from three replicates could be used to identify up- or down-modulated genes. Using this quantitative criteria, gene expression differences between two related T lymphoma cell lines, HuT78 and H9, were identified. The relevance of these findings to the known functional differences between these cell types is discussed.  相似文献   

14.
Functional protein microarrays for parallel characterisation of p53 mutants   总被引:1,自引:0,他引:1  
Understanding the way in which single nucleotide polymorphisms and mutations in the human genome result in individual susceptibility to disease is a major goal in the postgenomic era. Such knowledge should accelerate the development of personalised medicine in which drug treatment can specifically match an individual's genotype. High-throughput DNA sequencing is generating the initial information required, but new technologies are required that can rapidly characterise the phenotypic effects of the identified polymorphisms. For example, many thousands of allelic variants of the p53 gene have been described and are responsible for more than 50% of cancers, however few of the protein products have been functionally characterised. Here we have quantified in parallel the effects of mutations and polymorphisms on the DNA-binding function of the p53 oncoprotein using a protein microarray, allowing their subclassification according to functional effect. Protein-protein interactions between p53 variants and (i) a regulatory oncoprotein, (ii) a regulatory kinase resulting in on-chip phosphorylation, are also described, suggesting the more general utility of this high-throughput assay format.  相似文献   

15.
Li CR  Santoso S  Lo DD 《Cellular immunology》2007,250(1-2):40-54
T cell homeostatic proliferation occurs on transfer of T cells into lymphopenic recipients; transferred cells undergo several rounds of division in the absence of specific antigen stimulation. For a quantitative analysis of this phenomenon, we applied a mathematical method to describe proliferating T cells to match peak distributions from actual CFSE dilution data. For in vitro stimulation of T cells with anti-CD3/anti-CD28, our simulation confirmed a high proportion of cells entering cell cycle with a low proportion undergoing apoptosis. When applied to homeostatic proliferation, it described striking differences in CD4 and CD8 T cell proliferation rates, and accurately predicted that successive divisions were accompanied by higher rates of apoptosis, limiting the accumulation of proliferating cells. Thus, the presence of multiple CFSE dilution peaks cannot be considered equivalent to lymphocyte expansion. Finally, genetic effects were identified that may help explain links between homeostatic proliferation and autoimmunity.  相似文献   

16.
17.
Transfected cell microarrays are considered to be a breakthrough methodology for high-throughput and high-content functional genomics. Here, recent advances in the cell microarray field are reviewed, along with its potential to increase the speed of determining gene function. These advances, combined with an increasing number and diversity of gene perturbing systems, such as RNAi and ectopic gene expression, provide tools for expanding our understanding of biology at the systems level.  相似文献   

18.
Quantitative detection of microbial genes by using DNA microarrays   总被引:8,自引:0,他引:8  
To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.  相似文献   

19.
The quantitative analysis of signaling networks requires highly sensitive methods for the time-resolved determination of protein phosphorylation. For this reason, we developed a quantitative protein microarray that monitors the activation of multiple signaling pathways in parallel, and at high temporal resolution. A label-free sandwich approach was combined with near infrared detection, thus permitting the accurate quantification of low-level phosphoproteins in limited biological samples corresponding to less than 50,000 cells, and with a very low standard deviation of approximately 5%. The identification of suitable antibody pairs was facilitated by determining their accuracy and dynamic range using our customized software package Quantpro. Thus, we are providing an important tool to generate quantitative data for systems biology approaches, and to drive innovative diagnostic applications.  相似文献   

20.
Understanding the differences between microarray and RNA-Seq technologies for measuring gene expression is necessary for informed design of experiments and choice of data analysis methods. Previous comparisons have come to sometimes contradictory conclusions, which we suggest result from a lack of attention to the intensity-dependent nature of variation generated by the technologies. To examine this trend, we carried out a parallel nested experiment performed simultaneously on the two technologies that systematically split variation into four stages (treatment, biological variation, library preparation and chip/lane noise), allowing a separation and comparison of the sources of variation in a well-controlled cellular system, Saccharomyces cerevisiae. With this novel dataset, we demonstrate that power and accuracy are more dependent on per-gene read depth in RNA-Seq than they are on fluorescence intensity in microarrays. However, we carried out quantitative PCR validations which indicate that microarrays may demonstrate greater systematic bias in low-intensity genes than in RNA-seq.  相似文献   

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