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1.
Genome Res ; 31(4): 689-697, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33674351

RESUMO

Systematic delineation of complex biological systems is an ever-challenging and resource-intensive process. Single-cell transcriptomics allows us to study cell-to-cell variability in complex tissues at an unprecedented resolution. Accurate modeling of gene expression plays a critical role in the statistical determination of tissue-specific gene expression patterns. In the past few years, considerable efforts have been made to identify appropriate parametric models for single-cell expression data. The zero-inflated version of Poisson/negative binomial and log-normal distributions have emerged as the most popular alternatives owing to their ability to accommodate high dropout rates, as commonly observed in single-cell data. Although the majority of the parametric approaches directly model expression estimates, we explore the potential of modeling expression ranks, as robust surrogates for transcript abundance. Here we examined the performance of the discrete generalized beta distribution (DGBD) on real data and devised a Wald-type test for comparing gene expression across two phenotypically divergent groups of single cells. We performed a comprehensive assessment of the proposed method to understand its advantages compared with some of the existing best-practice approaches. We concluded that besides striking a reasonable balance between Type I and Type II errors, ROSeq, the proposed differential expression test, is exceptionally robust to expression noise and scales rapidly with increasing sample size. For wider dissemination and adoption of the method, we created an R package called ROSeq and made it available on the Bioconductor platform.


Assuntos
Perfilação da Expressão Gênica , RNA-Seq , Análise de Célula Única , Transcriptoma
2.
Commun Biol ; 5(1): 1231, 2022 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371461

RESUMO

Cell-cell communication and physical interactions play a vital role in cancer initiation, homeostasis, progression, and immune response. Here, we report a system that combines live capture of different cell types, co-incubation, time-lapse imaging, and gene expression profiling of doublets using a microfluidic integrated fluidic circuit that enables measurement of physical distances between cells and the associated transcriptional profiles due to cell-cell interactions. We track the temporal variations in natural killer-triple-negative breast cancer cell distances and compare them with terminal cellular transcriptome profiles. The results show the time-bound activities of regulatory modules and allude to the existence of transcriptional memory. Our experimental and bioinformatic approaches serve as a proof of concept for interrogating live-cell interactions at doublet resolution. Together, our findings highlight the use of our approach across different cancers and cell types.


Assuntos
Transcriptoma , Neoplasias de Mama Triplo Negativas , Humanos , Microfluídica , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica
3.
Electrophoresis ; 31(16): 2804-12, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20737447

RESUMO

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.


Assuntos
Bacteriólise/fisiologia , Esporos Bacterianos/fisiologia , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Bacillus/isolamento & purificação , Bacillus/fisiologia , Bacillus anthracis/genética , Bacillus anthracis/fisiologia , Bacillus cereus/genética , Bacillus cereus/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Ação Capilar , Divisão Celular , Primers do DNA , DNA Bacteriano/genética , Corantes Fluorescentes , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Solubilidade , Esporos Bacterianos/genética , Esporos Bacterianos/isolamento & purificação , Termodinâmica
5.
Artigo em Inglês | MEDLINE | ID: mdl-27709111

RESUMO

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.

6.
J Lab Autom ; 16(5): 355-65, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21906561

RESUMO

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.


Assuntos
Automação Laboratorial/instrumentação , Automação Laboratorial/métodos , Ácidos Nucleicos/isolamento & purificação , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Bactérias/genética , Sangue/microbiologia , Sangue/virologia , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Plasma/virologia , Vírus/genética , Microbiologia da Água
7.
Anal Chem ; 80(23): 9005-12, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19551975

RESUMO

We present a rapid method for the identification of viruses using microfluidic chip gel electrophoresis (CGE) of high-copy number proteins to generate unique protein profiles. Viral proteins are solubilized by heating at 95 degrees C in borate buffer containing detergent (5 min), then labeled with fluorescamine dye (10 s), and analyzed using the microChemLab CGE system (5 min). Analyses of closely related T2 and T4 bacteriophage demonstrate sufficient assay sensitivity and peak resolution to distinguish the two phage. CGE analyses of four additional viruses--MS2 bacteriophage, Epstein-Barr, respiratory syncytial, and vaccinia viruses--demonstrate reproducible and visually distinct protein profiles. To evaluate the suitability of the method for unique identification of viruses, we employed a Bayesian classification approach. Using a subset of 126 replicate electropherograms of the six viruses and phage for training purposes, successful classification with non-training data was 66/69 or 95% with no false positives. The classification method is based on a single attribute (elution time), although other attributes such as peak width, peak amplitude, or peak shape could be incorporated and may improve performance further. The encouraging results suggest a rapid and simple way to identify viruses without requiring specialty reagents such as PCR probes and antibodies.


Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Proteínas Virais/análise , Vírus/química , Bacteriófagos/química , Calibragem , Eletroforese em Microchip/economia , Eletroforese em Gel de Poliacrilamida , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/economia , Sensibilidade e Especificidade , Fatores de Tempo
8.
Anal Chem ; 79(16): 6230-5, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17625914

RESUMO

Efficient and rapid isolation of mRNA is important in the field of genomics as well as in the clinical and pharmaceutical arena. We have developed UV-initiated methacrylate-based porous polymer monoliths (PPM) for microfluidic trapping and concentration of eukaryotic mRNA. PPM are cast-to-shape and are tunable for functionalization using a variety of amine-terminated molecules. Efficient isolation of eukaryotic mRNA from total RNA was first mathematically modeled and then achieved using PPM in capillaries. Purification protocols using oligo dT's, locked nucleic acid substituted dT's, and tetramethylammonium chloride salts were characterized. mRNA yield and purity were compared with mRNA isolated by commercial kits with statistically equivalent yields and purities (determined by qPCR ratio of 18s rRNA and Gusb mRNA markers). Even after extracting 16 microg of mRNA from 315 microg of total RNA, the 0.4-microL volume monolith showed no signs of saturation. Elution volumes were below 20 microL with concentrations up to 1 microg/microL. In addition, the polymeric material exhibited exceptional stability in a range of conditions (pH, temperature, dryness) and was stable for a period of months. All of these characteristics make porous polymer monoliths good candidates for potential microfluidic sample preconcentrators and purifiers.


Assuntos
Microfluídica/instrumentação , Microfluídica/métodos , Polímeros , RNA Mensageiro/isolamento & purificação , Metacrilatos , Porosidade
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