Spatially resolved and multiplexed MicroRNA quantification from tissue using nanoliter well arrays.
Ontology highlight
ABSTRACT:
SUBMITTER: Nagarajan MB
PROVIDER: S-EPMC7211184 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:MicroRNAs (miRNAs) have recently emerged as promising biomarkers for the profiling of diseases. Translation of miRNA biomarkers to clinical practice, however, remains a challenge due to the lack of analysis platforms for sensitive, quantitative, and multiplex miRNA assays that have simple and robust workflows suitable for translation. The platform we present here utilizes functionalized hydrogel posts contained within isolated nanoliter well reactors for quantitative and multiplex assays directly from unprocessed cell samples without the need of prior nucleic acid extraction. Simultaneous reactor isolation and delivery of miRNA extraction reagents is achieved by sealing an array of wells containing the functionalized hydrogel posts and cells against another array of wells containing lysis and extraction reagents. The nanoliter well array platform features >100× better sensitivity compared to previous technology utilizing hydrogel particles without relying on signal amplification and enables >100 parallel assays in a single device. These advances provided by this platform lay the groundwork for translatable and robust analysis technologies for miRNA expression profiling in samples with small populations of cells and in precious, material-limited samples.
| S-EPMC6081239 | biostudies-literature
Project description:Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 – 1000 unique RNA species in hundreds of individual cells. Correlation analysis of the ~10^4 – 10^6 pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins. A single sample is analyzed
2015-04-09 | E-GEOD-67685 | biostudies-arrayexpress
Project description:Antibiotic resistance is a major global health concern that requires action across all sectors of society. In particular, to allow conservative and effective use of antibiotics clinical settings require better diagnostic tools that provide rapid determination of antimicrobial susceptibility. We present a method for rapid and scalable antimicrobial susceptibility testing using stationary nanoliter droplet arrays that is capable of delivering results in approximately half the time of conventional methods, allowing its results to be used the same working day. In addition, we present an algorithm for automated data analysis and a multiplexing system promoting practicality and translatability for clinical settings. We test the efficacy of our approach on numerous clinical isolates and demonstrate a 2-d reduction in diagnostic time when testing bacteria isolated directly from urine samples.
| S-EPMC5530678 | biostudies-literature
Project description:Parallelized quantum information processing requires tailored quantum memories to simultaneously handle multiple photons. The spatial degree of freedom is a promising candidate to facilitate such photonic multiplexing. Using a single-photon resolving camera, we demonstrate a wavevector multiplexed quantum memory based on a cold atomic ensemble. Observation of nonclassical correlations between Raman scattered photons is confirmed by an average value of the second-order correlation function [Formula: see text] in 665 separated modes simultaneously. The proposed protocol utilizing the multimode memory along with the camera will facilitate generation of multi-photon states, which are a necessity in quantum-enhanced sensing technologies and as an input to photonic quantum circuits.
| S-EPMC5732182 | biostudies-other
Project description:Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~10(4) to 10(6) pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.
| S-EPMC4662681 | biostudies-literature
Project description:Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 – 1000 unique RNA species in hundreds of individual cells. Correlation analysis of the ~10^4 – 10^6 pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.
2015-04-09 | GSE67685 | GEO
Project description:There is great demand for flexible biomolecule analysis platforms that can precisely quantify very low levels of multiple targets directly in complex biological samples. Herein we demonstrate multiplexed quantification of microRNAs (miRNAs) on encoded hydrogel microparticles with subfemtomolar sensitivity and single-molecule reporting resolution. Rolling circle amplification (RCA) of a universal adapter sequence that is ligated to all miRNA targets captured on gel-embedded probes provides the ability to label each target with multiple fluorescent reporters and eliminates the possibility of amplification bias. The high degree of sensitivity achieved by the RCA scheme and the resistance to fouling afforded by the use of gel particles are leveraged to directly detect miRNA in small quantities of unprocessed human serum samples without the need for RNA extraction or target-amplification steps. This versatility has powerful implications for the development of rapid, noninvasive diagnostic assays.
| S-EPMC3173568 | biostudies-literature
Project description:The lymph node is a structurally complex organ of the immune system, whose dynamic cellular arrangements are thought to control much of human health. Currently, no methods exist to precisely stimulate substructures within the lymph node or analyze local stimulus-response behaviors, making it difficult to rationally design therapies for inflammatory disease. Here we describe a novel integration of live lymph node slices with a microfluidic system for local stimulation. Slices maintained the cellular organization of the lymph node while making its core experimentally accessible. The 3-layer polydimethylsiloxane device consisted of a perfusion chamber stacked atop stimulation ports fed by underlying microfluidic channels. Fluorescent dextrans similar in size to common proteins, 40 and 70 kDa, were delivered to live lymph node slices with 284 ± 9 μm and 202 ± 15 μm spatial resolution, respectively, after 5 s, which is sufficient to target functional zones of the lymph node. The spread and quantity of stimulation were controlled by varying the flow rates of delivery; these were predictable using a computational model of isotropic diffusion and convection through the tissue. Delivery to two separate regions simultaneously was demonstrated, to mimic complex intercellular signaling. Delivery of a model therapeutic, glucose-conjugated albumin, to specific regions of the lymph node indicated that retention of the drug was greater in the B-cell zone than in the T-cell zone. Together, this work provides a novel platform, the lymph node slice-on-a-chip, to target and study local events in the lymph node and to inform the development of new immunotherapeutics.
| S-EPMC7863610 | biostudies-literature
Project description:We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-mum-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 microm) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of high-density individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight spaces, as well as for developing multifunctional electrochemical microsensors.
| S-EPMC2653425 | biostudies-other
Project description:The SlipChip platform was tested to perform high-throughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and high-throughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization-based arrays.
| S-EPMC2916686 | biostudies-literature