Multiplexed direct detection of barcoded protein reporters on a nanopore array.
Ontology highlight
ABSTRACT:
SUBMITTER: Cardozo N
PROVIDER: S-EPMC8766897 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Magnetic polymer particles have been used in a wide variety of applications ranging from targeting and separation to diagnostics and imaging. Current synthesis methods have limited these particles to spherical or deformations of spherical morphologies. In this paper, we report the use of stop flow lithography to produce magnetic hydrogel microparticles with a graphical code region, a probe region, and a magnetic tail region. These anisotropic multifunctional magnetic polymer particles are an enhanced version of previously synthesized "barcoded" particles (Science, 2007, 315, 1393-1396) developed for the sensitive and rapid multiplexed sensing of nucleic acids. The newly added magnetic region has acquired dipole moments in the presence of weak homogeneous magnetic fields, allowing the particles to align along the applied field direction. The novel magnetic properties have led to practical applications in the efficient orientation and separation of the barcoded microparticles during biological assays without disrupting detection capabilities.
| S-EPMC2877154 | biostudies-literature
Project description:MicroRNAs are a class of small RNA molecules that regulate the expression of mRNAs in a wide range of biological processes and are implicated in human health and disease such as cancers. How to measure microRNA profiles in single cells with high throughput is essential to the development of cell-based assays for interrogating microRNA-mediated intratumor heterogeneity and the design of new lab tests for diagnosis and monitoring of cancers. Here, we report on an in situ hybridization barcoding workflow implemented in a sub-nanoliter microtrough array chip for high-throughput and multiplexed microRNA detection at the single cell level. The microtroughs are used to encapsulate single cells that are fixed, permeabilized, and pre-incubated with microRNA detection probes, each of which consists of a capture strand complementary to specific microRNA and a unique reporter strand that can be photocleaved in the microtroughs and subsequently detected by an array of DNA barcodes patterned on the bottom of the microtroughs. In this way, the measurement of reporter strands released from single cells is a surrogate for detecting single-cell microRNA profiles. This approach permits direct measurement of microRNAs without PCR amplification owing to the small volume (<1 nL) of microtroughs. It offers high throughput and high multiplexing capability for evaluating microRNA heterogeneity in single cells, representing a new approach toward microRNA-based diagnosis and monitoring of complex human diseases.
| S-EPMC6523668 | biostudies-literature
Project description:We demonstrate the use of graphically encoded hydrogel microparticles for the sensitive and high-throughput multiplexed detection of clinically relevant protein panels in complex media. Combining established antibody capture techniques with advances in both microfluidic synthesis and analysis, we detected 1-8 pg/mL amounts of three cytokines (interleuken-2, interleuken-4, and tumor necrosis factor alpha) in single and multiplexed assays without the need for filtration or blocking agents. A range of hydrogel porosities was investigated to ensure rapid diffusion of targets and reagents into the particle as well as to maintain the structural integrity of particles during rinsing procedures and high-velocity microfluidic scanning. Covalent incorporation of capture antibodies using a heterobifunctional poly(ethylene glycol) linker enabled one-step synthesis and functionalization of particles using only small amounts of valuable reagents. In addition to the use of three separate types of single-probe particles, the flexibility of the stop-flow lithography (SFL) method was leveraged to spatially segregate the three probes for the aforementioned target set on an individual encoded particle, thereby demonstrating the feasibility of single-particle diagnostic panels. This study establishes the gel-particle platform as a versatile tool for the efficient quantification of protein targets and significantly advances efforts to extend the advantages of both hydrogel substrates and particle-based arrays to the field of clinical proteomics.
| S-EPMC3030995 | biostudies-literature
Project description:In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance in determining cell identity and their pivotal role in numerous disorders, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 60 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways.
| S-EPMC11291157 | biostudies-literature
Project description:A library of 35,500 reporters was designed for 86 TFs in order to identify optimized TF reporters. The library was transfected into diverse cell types and probed upon almost 100 TF perturbations. To compare TF activities with TF abundances, RNA-seq data was used. For mNPCs, mESCs and HepG2 cells, RNA-seq data was generated in house. For thee TF knockdowns in HepG2 cells, RNA-seq data was generated to validate the experimental design.
2024-05-26 | GSE267969 | GEO
Project description:Cis-regulatory DNA sequences causally mediate patterns of gene expression, but efficient experimental analysis of these control systems has remained challenging. Here we develop a new version of "barcoded" DNA-tag reporters, "Nanotags" that permit simultaneous quantitative analysis of up to 130 distinct cis-regulatory modules (CRMs). The activities of these reporters are measured in single experiments by the NanoString RNA counting method and other quantitative procedures. We demonstrate the efficiency of the Nanotag method by simultaneously measuring hourly temporal activities of 126 CRMs from 46 genes in the developing sea urchin embryo, otherwise a virtually impossible task. Nanotags are also used in gene perturbation experiments to reveal cis-regulatory responses of many CRMs at once. Nanotag methodology can be applied to many research areas, ranging from gene regulatory networks to functional and evolutionary genomics.
| S-EPMC3339872 | biostudies-literature
Project description:Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.
| S-EPMC5856557 | biostudies-literature
Project description:The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas-based knockouts and RNA-interference-based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.
| S-EPMC4780610 | biostudies-literature
Project description:Highly multiplexed detection of proteins secreted by single cells is always challenging. Herein, a multiplexed in situ tagging technique based on single-stranded DNA encoded microbead arrays and multicolor successive imaging for assaying single-cell secreted proteins with high throughput and high sensitivity is presented. This technology is demonstrated to be capable of increasing the multiplexity exponentially. Upon integration with polydimethylsiloxane microwells, this platform is applied to detect ten immune effector proteins from differentiated single macrophages stimulated with lipopolysaccharide. Significant heterogeneity is observed when the derived human primary macrophages are analyzed. This versatile technology is expected to open new opportunities in systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.
| S-EPMC6145231 | biostudies-literature
Project description:Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.
| S-EPMC11347153 | biostudies-literature