Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Eukaryotic Cell Capture by Amplified Magnetic in situ Hybridization Using Yeast as a Model

ABSTRACT: A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.

SUBMITTER: Bastian F

PROVIDER: S-EPMC8591292 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Targeted DNA methylation from cell-free DNA using hybridization probe capture.

Project description:Cell-free (cf)DNA signatures are quickly becoming the target of choice for non-invasive screening, diagnosis, treatment and monitoring of human tumors. DNA methylation changes occur early in tumorigenesis and are widespread, making cfDNA methylation an attractive cancer biomarker. Already a proven technology for targeted genome sequencing, hybridization probe capture is emerging as a method for high-throughput targeted methylation profiling suitable to liquid biopsy samples. However, to date there are no reports describing the performance of this approach in terms of reproducibility, scalability, and accuracy. In the current study we performed hybridization probe capture using the myBaits® Custom Methyl-seq kit on 172 plasma samples and standards to evaluate its performance on cfDNA methylation analysis. The myBaits® assay showed high target recovery (>90%), demonstrated excellent reproducibility between captures (R 2 = 0.92 on average), and was unaffected by increasing the number of targets in a capture. Finally, myBaits® accurately replicated 'gold standard' beta values from WGBS (average R 2 = 0.79). The results of this study show that custom targeted methylation sequencing with myBaits® offers a cost-effective, reliable platform to profile DNA methylation at a set of discrete custom regions, with potential applicability to liquid biopsies for cancer monitoring.

| S-EPMC9803870 | biostudies-literature

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.

Project description:Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.

| S-EPMC4265680 | biostudies-literature

Revealing microbial species diversity using sequence capture by hybridization.

Project description:Targeting small parts of the 16S rDNA phylogenetic marker by metabarcoding reveals microorganisms of interest but cannot achieve a taxonomic resolution at the species level, precluding further precise characterizations. To identify species behind operational taxonomic units (OTUs) of interest, even in the rare biosphere, we developed an innovative strategy using gene capture by hybridization. From three OTU sequences detected upon polyphenol supplementation and belonging to the rare biosphere of the human gut microbiota, we revealed 59 nearly full-length 16S rRNA genes, highlighting high bacterial diversity hidden behind OTUs while evidencing novel taxa. Inside each OTU, revealed 16S rDNA sequences could be highly distant from each other with similarities down to 85 %. We identified one new family belonging to the order Clostridiales, 39 new genera and 52 novel species. Related bacteria potentially involved in polyphenol degradation have also been identified through genome mining and our results suggest that the human gut microbiota could be much more diverse than previously thought.

| S-EPMC8767324 | biostudies-literature

Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry.

Project description:Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.

| S-EPMC4350577 | biostudies-literature

Hybridization capture reveals microbial diversity missed using current profiling methods.

Project description:BACKGROUND:Microorganisms comprise the majority of living organisms on our planet. For many years, exploration of the composition of microbial communities has been performed through the PCR-based study of the small subunit rRNA gene due to its high conservation across the domains of life. The application of this method has resulted in the discovery of many unexpected evolutionary lineages. However, amplicon sequencing is subject to numerous biases, with some taxa being missed, and is limited by the read length of second-generation sequencing platforms, which drastically reduces the phylogenetic resolution. RESULTS:Here, we describe a hybridization capture strategy that allows the enrichment of 16S rRNA genes from metagenomic samples and enables an exhaustive identification and a complete reconstruction of the biomarker. Applying this approach to a microbial mock community and a soil sample, we demonstrated that hybridization capture is able to reveal greater microbial diversity than 16S rDNA amplicon sequencing and shotgun sequencing. The reconstruction of full-length 16S rRNA genes facilitated the improvement of phylogenetic resolution and the discovery of novel prokaryotic taxa. CONCLUSIONS:Our results demonstrate that hybridization capture can lead to major breakthroughs in our understanding of microbial diversity, overcoming the limitations of conventional 16S rRNA gene studies. If applied to a broad range of environmental samples, this innovative approach could reveal the undescribed diversity of the still underexplored microbial communities and could provide a better understanding of ecosystem function.

| S-EPMC5870382 | biostudies-literature

Scalable in situ single-cell profiling by electrophoretic capture of mRNA using EEL FISH.

Project description:Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here we develop a method named Enhanced ELectric Fluorescence in situ Hybridization (EEL FISH) that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down toward the surface, preserving single-cell resolution. We apply EEL on eight entire sagittal sections of the mouse brain and measure the expression patterns of up to 440 genes to reveal complex tissue organization. Moreover, EEL can be used to study challenging human samples by removing autofluorescent lipofuscin, enabling the spatial transcriptome of the human visual cortex to be visualized. We provide full hardware specifications, all protocols and complete software for instrument control, image processing, data analysis and visualization.

| S-EPMC9931581 | biostudies-literature

Validation of terminal Schwann cell gene marker expression by fluorescent in situ hybridization using RNAscope.

Project description:Recent RNA-seq studies have generated a new crop of putative gene markers for terminal Schwann cells (tSCs), non-myelinating glia that cap axon terminals at the vertebrate neuromuscular junction (NMJ). While compelling, these studies did not validate the expression of the novel markers using in situ hybridization techniques. Here, we use RNAscope technology to study the expression of top candidates from recent tSC and non-myelinating Schwann cell marker RNA-seq studies. Our results validate the expression of these markers at tSCs but also demonstrate that they are present at other sites in the muscle tissue, specifically, at muscle spindles and along intramuscular nerves.

| S-EPMC8821205 | biostudies-literature

Classification of cancer cell lines using a promoter-targeted liquid hybridization capture-based bisulfite sequencing approach

Project description:DNA methylation plays a significant role in assuring cell identity, thus potentiating its application in molecular classification of cancers in respect of tissue origins or clinically and aetiologically distinct subtypes. In this study, we adapted our liquid hybridization capture-based bisulfite sequencing approach on the targeted sequencing of promoter methylomes. We detected ten cell lines originated from different tissue origins and demonstrated a similar potentiality of promoter methylomes as classifiers for cancer cell lines from different tissue origins in comparison with gene expression profiles. Furthermore, promoter methylome can sensitively differentiate two different cell lines from the same tissue origin in respect of the CpG island methylator phenotype (CIMP), as in the case of AGS and BGC-823 gastric cancer cell lines. These results potentiate the targeted sequencing of promoter methylomes as a means for comprehensive screening and classifying cancer cells with respect to tissue-origins and CIMP subtypes in the future studies. We proved the potentiality of promoter-targeted LHC-BS that requires reduced experimental cost and less amount of initial DNA samples in comparison with a previous design [23] using YH cell line. In addition, we generated single-base promoter methylomes for ten cell lines, including eight cancer cell lines generated from four types of tissues and one pair of model cell lines for ovarian cancer (T29 and T29H). We proved the potentiality of promoter-targeted LHC-BS that requires reduced experimental cost and less amount of initial DNA samples in comparison with a previous design using YH cell line. In addition, we generated single-base promoter methylomes for ten cell lines, including eight cancer cell lines generated from four types of tissues and one pair of model cell lines for ovarian cancer (T29 and T29H).

| S-ECPF-GEOD-44866 | biostudies-other

Improved Equine Fecal Microbiome Characterization Using Target Enrichment by Hybridization Capture.

Project description:GITDs are among the most common causes of death in adult and young horses in the United States (US). Previous studies have indicated a connection between GITDs and the equine gut microbiome. However, the low taxonomic resolution of the current microbiome sequencing methods has hampered the identification of specific bacterial changes associated with GITDs in horses. Here, we have compared TEHC, a new approach for 16S rRNA gene selection and sequencing, with conventional 16S rRNA gene amplicon sequencing for the characterization of the equine fecal microbiome. Both sequencing approaches were used to determine the fecal microbiome of four adult horses and one commercial mock microbiome. Our results show that TEHC yielded significantly more operational taxonomic units (OTUs) than conventional 16S amplicon sequencing when the same number of reads were used in the analysis. This translated into a deeper and more accurate characterization of the fecal microbiome when the samples were sequenced with TEHC according to the relative abundance analysis. Alpha and beta diversity metrics corroborated these findings and demonstrated that the microbiome of the fecal samples was significantly richer when sequenced with TEHC compared to 16S amplicon sequencing. Altogether, our study suggests that the TEHC strategy provides a more extensive characterization of the fecal microbiome of horses than the current alternative based on the PCR amplification of a portion of the 16S rRNA gene.

| S-EPMC10854943 | biostudies-literature

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Project description:Though important for gene regulation most studies of genome organisation use either fluorescence in situ hybridisation (FISH) or chromosome conformation capture (3C) methods. FISH directly visualises the spatial relationship of sequences, but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde crosslinking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organisation, but this has not been tested extensively. We investigate the murine HoxD locus with 5C and FISH in different developmental and activity states, and in the presence or absence of epigenetic regulators. We identify situations where the two datasets are concordant, but find other conditions where chromatin topographies extrapolated from 5C or FISH data are not compatible. We conclude that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart – influenced by nuclear environment and chromatin composition. We conclude that results obtained at high-resolution with either 3C methods or by FISH alone must be interpreted with caution and that conclusions about genome organisation should be validated by independent methods.

2015-01-02 | GSE61814 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data