| 1. |
- Irimia, Daniel, et al.
(författare)
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Genome-wide transcriptome analysis of 150 cell samples
- 2009
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Ingår i: Integrative Biology. - : Oxford University Press (OUP). - 1757-9694 .- 1757-9708. ; 1:1, s. 99-107
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Tidskriftsartikel (refereegranskat)abstract
- major challenge in molecular biology is interrogating the human transcriptome on a genome wide scale when only a limited amount of biological sample is available for analysis. Current methodologies using microarray technologies for simultaneously monitoring mRNA transcription levels, require nanogram amounts of total RNA. To overcome the sample size limitation of Current technologies, we have developed a method to probe the global gene expression ill biological samples as small as 150 cells, or the equivalent of approximately 300 pg total RNA. The new method employs microfluidic devices for the purification of total RNA from mammalian cells and ultra-sensitive whole transcriptome amplification techniques. We verified that the RNA integrity is preserved through the isolation process, accomplished highly reproducible whole transcriptome analysis, and established high correlation between repeated isolations of 150 cells and the same cell culture sample. We validated the technology by demonstrating that the combined microfluidic and amplification protocol is capable of identifying biological pathways perturbed by Stimulation, which are consistent with the information recognized in bulk-isolated samples.
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| 2. |
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| 3. |
- Kotz, Kenneth T., et al.
(författare)
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Clinical microfluidics for neutrophil genomics and proteomics
- 2010
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 16:9, s. 1042-U142
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.
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| 4. |
- Lindström, Sara, 1980-
(författare)
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Microwell devices for single-cell analyses
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.
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| 5. |
- Russom, Aman, et al.
(författare)
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Chemical gradient-mediated melting curve analysis for genotyping of SNPs
- 2009
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 30:14, s. 2536-2543
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Tidskriftsartikel (refereegranskat)abstract
- This report describes a microfluidic solid-phase chemical gradient-mediated melting curve analysis method for SNP analysis. The method is based on allele-specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson-Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP.
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| 6. |
- Russom, Aman, et al.
(författare)
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Differential inertial focusing of particles in curved low-aspect-ratio microchannels
- 2009
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Ingår i: New Journal of Physics. - : IOP Publishing. - 1367-2630. ; 11, s. 075025-
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Tidskriftsartikel (refereegranskat)abstract
- Microfluidic-based manipulation of particles is of great interest due to the insight it provides into the physics of hydrodynamic forces. Here, we study a particle-size-dependent phenomenon based on differential inertial focusing that utilizes the flow characteristics of curved, low aspect ratio (channel width >> height), microfluidic channels. We report the emergence of two focusing points along the height of the channel (z-plane), where different sized particles are focused and ordered in evenly spaced trains at correspondingly different lateral positions within the channel cross-section. We applied the system for continuous ordering and separation of suspension particles.
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| 7. |
- Russom, Aman, et al.
(författare)
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Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:5, s. 891-900
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. METHODS: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. RESULTS: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. CONCLUSIONS: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting. (c) 2008 American Association for Clinical Chemistry.
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| 8. |
- Toner, Mehmet, et al.
(författare)
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Systems and methods for particle focusing in microchannels
- 2007
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Patent (populärvet., debatt m.m.)abstract
- Novelty: A biological system comprises substrate, channels (16) provided on substrate, fluid moving along channel in laminar flow and including suspended particles (20), pumping element driving laminar flow of fluid, detector, tagging system. The fluid, channel, and pumping element are configured to cause inertial forces to act on particles and to focus particles into stream lines. The channel has hydraulic diameter. Moving the fluid suspension focuses particles into localized streams. Location of focused stream channel depends on inertial forces and Dean drag forces acting on particles.Use: A system for focusing, sorting, and separating target particles (claimed) useful in industrial application including chemical process and fermentation filtration, water purification and wastewater treatment; in biomedical application including counting, sorting and filtering blood components and preparing safely sized micro-bubble ultrasound contrast agents; in research including tissue engineering, drug control release mechanism studies, cell signaling studies, protein crystallization, virus/bacteria capture, nucleic acid purification, and chemistry specific extractions; in the development of cosmetics, lubricants, pigments, environmental monitoring for particulates, natural oil extraction, particle synthesis, and polymer bead manufacturing; and in agriculture including development of multi-phase fertilizer emulsions, multi-phase pesticide emulsions, flow cytometry, and in hematology analysis.Advantage: The system can perform rapid continuous processing of samples without the need for filters or mechanical or electrical parts, high throughput applications, low noise results, and independence in focusing for particle shape and density. The inertial focusing of the system results in precise particle positioning into single stream and controlled longitudinal spacing between particles. Precise control of particle streamlines allows sorting with small induced changes in particle position. The system has the ability to perform cell ordering and separation at the point of care without the need for bulky equipment. The system also creates opportunities for rapid screening of patients for a number of diseases.
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