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Dataset results
19 results for “Fluorescent In Situ Hybridization”
Single-molecule Fluorescent In Situ Hybridization (smFISH) for RNA detection in the fungal pathogen Candida albicans dataset
<p><strong>This dataset is connected to the protocol article titled:</strong></p> <p>Single-molecule Fluorescent <em>In Situ</em> Hybridization (smFISH) for RNA detection in the fungal pathogen <em>Candida albicans</em></p> <p><strong>Abstract:</strong></p> <p><em>Candida albicans</em> is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of <em>C. albicans</em> to reversibly change morphology and to grow as yeast, pseudohyphal or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help fight <em>C. albicans</em> infections.</p> <p>While numerous studies investigated gene expression changes occurring during <em>C. albicans</em> morphological switches using bulk approaches (e.g., RNA sequencing), here we describe a single-cell and single-molecule RNA imaging and analysis protocol to measure absolute mRNA counts in morphologically intact cells. To detect endogenous mRNAs in single fixed cells, we optimized a single molecule fluorescent <em>in situ</em> hybridization (smFISH) protocol for <em>C. albicans</em>, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae or hyphal cells. We quantified the expression of two mRNAs, cell cycle-controlled mRNA (<em>CLB2)</em> and a transcription regulator (<em>EFG1</em>), which show differential expression in the different morphological cell types and in different nutrient conditions. In this protocol we described in detail the major steps of this approach: growth and fixation, hybridization, imaging, cell-segmentation and mRNA spot analysis. Raw data is provided with the protocol to favour reproducibility. This approach could benefit the molecular characterization of <em>C. albicans</em> and other filamentous fungi, pathogenic or non-pathogenic.</p> <p><strong>Data description:</strong></p> <p>This dataset consists of a FISH experiment spanning two different mRNAs, EFG1 and CLB2, and two different nutrient condition, being SPIDER37 and TSB37 in Candida albicans. For culturing, the C. albicans wildtype strain SC5314 was inoculated at 30 degrees overnight (~15 hours) in 10 mL of TSB medium in a 30 degree (celsius) shaking incubator. Next, samples were diluted to a density of 10^5 cells/ mL and inoculated for 6 hours in either 30 mL TSB medium or Spider medium at 37 degrees in falcon tubes on an orbital microplate shaker. Then, samples were fixated by adding PFA to a final concentration of 4% to the medium. For hybridization, both mRNAs were hybridized independently by specific DNA oligo labelled with a Quasar670 dye to enable the visualisation of single mRNA molecules. As both genes are labelled by the same dye, these oligos were not co-applied to the same sample but to independent samples.</p> <p><strong>Microscopy</strong></p> <p>For smFISH imaging we use an Olympus BX-63 epifluorescence microscope equipped with Ultrasonic stage and UPlanApo 100x 1.35NA oil-immersion objective (Olympus). Lumencore SOLA FISH light source, a Hamamatsu ORCA-Fusion sCMOS camera (6.5 µm-pixel size) mounted using U-CMT C-Mount Adapter, and zero-pixel shift filter sets: F36-500 DAPI HC Brightline Bandpass Filter, F36-502 FITC HC BrightLine Filter, F36-542 Cy3 HC BrightLine Filter, and F36-523 Cy5 HC BrightLine Filter. Images are acquired across 61-81 optical sections (depending on the sample thickness) with a z-step size of 0.2 μm. The CellSens software (Olympus) is used for instrument control and image acquisition. For the DAPI channel 10-50 ms of exposure was used. Whilst, for the CY5 channel, used imaging the FISH probes, 750 ms was applied. </p> <p> </p>
Single-molecule Fluorescent In Situ Hybridization (smFISH) for RNA detection in the fungal pathogen Candida albicans small example dataset
<p><strong>This small example dataset is connected to the protocol article titled:</strong></p> <p>Single-molecule Fluorescent <em>In Situ</em> Hybridization (smFISH) for RNA detection in the fungal pathogen <em>Candida albicans</em></p> <p><strong>Abstract:</strong></p> <p><em>Candida albicans</em> is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of <em>C. albicans</em> to reversibly change morphology and to grow as yeast, pseudohyphal or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help fight <em>C. albicans</em> infections.</p> <p>While numerous studies investigated gene expression changes occurring during <em>C. albicans</em> morphological switches using bulk approaches (e.g., RNA sequencing), here we describe a single-cell and single-molecule RNA imaging and analysis protocol to measure absolute mRNA counts in morphologically intact cells. To detect endogenous mRNAs in single fixed cells, we optimized a single molecule fluorescent <em>in situ</em> hybridization (smFISH) protocol for <em>C. albicans</em>, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae or hyphal cells. We quantified the expression of two mRNAs, cell cycle-controlled mRNA (<em>CLB2)</em> and a transcription regulator (<em>EFG1</em>), which show differential expression in the different morphological cell types and in different nutrient conditions. In this protocol, we described in detail the major steps of this approach: growth and fixation, hybridization, imaging, cell-segmentation and mRNA spot analysis. Raw data is provided with the protocol to favour reproducibility. This approach could benefit the molecular characterization of <em>C. albicans</em> and other filamentous fungi, pathogenic or non-pathogenic.</p> <p><strong>Data description:</strong></p> <p>This dataset consists of a FISH experiment spanning two different mRNAs, EFG1 and CLB2, and one nutrient condition, SPIDER37, in Candida albicans. For culturing, the C. albicans wildtype strain SC5314 was inoculated at 30 degrees overnight (~15 hours) in 10 mL of TSB medium in a 30 °C shaking incubator. Next, samples were diluted to a density of 10^5 cells/ mL and inoculated for 6 hours in 30 mL Spider medium at 37 °C in falcon tubes on an orbital microplate shaker. Then, samples were fixated by adding PFA to a final concentration of 4% to the medium. For hybridization, both mRNAs were hybridized independently by specific DNA oligo labelled with a Quasar670 dye to enable the visualisation of single mRNA molecules. As both genes are labelled by the same dye, these oligos were not co-applied to the same sample but to independent samples.</p> <p><strong>Microscopy</strong></p> <p>For smFISH imaging we use an Olympus BX-63 epifluorescence microscope equipped with Ultrasonic stage and UPlanApo 100x 1.35NA oil-immersion objective (Olympus). Lumencore SOLA FISH light source, a Hamamatsu ORCA-Fusion sCMOS camera (6.5 µm-pixel size) mounted using U-CMT C-Mount Adapter, and zero-pixel shift filter sets: F36-500 DAPI HC Brightline Bandpass Filter, F36-502 FITC HC BrightLine Filter, F36-542 Cy3 HC BrightLine Filter, and F36-523 Cy5 HC BrightLine Filter. Images are acquired across 61-81 optical sections (depending on the sample thickness) with a z-step size of 0.2 μm. The CellSens software (Olympus) is used for instrument control and image acquisition. For the DAPI channel 10-50 ms of exposure was used. Whilst, for the CY5 channel, used for imaging the FISH probes, 750 ms was applied. </p>
Reference genes for quantitative Arabidopsis single molecule RNA fluorescence in situ hybridization
Abstract Subcellular mRNA quantities and spatial distributions are fundamental for driving gene regulatory programmes. Single molecule RNA fluorescence in situ hybridization (smFISH) uses fluorescent probes to label individual mRNA molecules, thereby facilitating both localization and quantitative studies. Validated reference mRNAs function as positive controls and are required for calibration. Here we present selection criteria for the first set of Arabidopsis smFISH reference genes. Following sequence and transcript data assessments, four mRNA probe sets were selected for imaging. Transcript counts per cell, correlations with cell size, and corrected fluorescence intensities were all calculated for comparison. In addition to validating reference probe sets, we present sample preparation steps that can retain green fluorescent protein fluorescence, thereby providing a method for simultaneous RNA and protein detection. In summary, our reference gene analyses, modified protocol, and simplified quantification method together provide a firm foundation for future quantitative single molecule RNA studies in Arabidopsis root apical meristem cells.
Example Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) data from the mouse colon
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Reference genes for quantitative Arabidopsis single molecule RNA fluorescence in situ hybridization
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Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes - Gutnik et al - Tracing Datasets
<p>Tracing datasets (MATLAB Structure Format) from <i>C.elegans</i> N2 and HI embryos, as well as N2:HI and HI:N2 hybrid embryos presented in Gutnik et al.2024 (<strong>Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal </strong><i><strong>C. elegans</strong></i><strong> chromosomes)</strong></p><p> </p><p> </p><p> </p>
Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes - Gutnik et al - Raw Imaging data
<p>Raw Imaging data for all figures presented in Gutnik et al.2024 (<strong>Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal </strong><i><strong>C. elegans</strong></i><strong> chromosomes)</strong></p>
Telomeric Abnormalities in Colorectal Diseases by Fluorescent in Situ Hybridization Technique
ClinicalTrials.gov study NCT03208777. IPD Sharing: NO. Countries: 0. Publications: 1.
Fluorescent in situ hybridization of Mycobacterium tuberculosis rRNA species for analysis of ribosomal synthesis as a marker of bacterial growth of individual bacilli in distinct lesional microenvironments of lung granulomas from C3HeB/FeJ mice
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Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization
GEO Series GSE61814. Mus musculus. 18 samples. Type: Other.
Multicolor miRNA fluorescence in situ hybridization for tumor differential diagnosis
GEO Series GSE34137. Homo sapiens. 40 samples. Type: Non-coding RNA profiling by high throughput sequencing.
Multicolor miRNA fluorescence in situ hybridization for tumor differential diagnosis
GEO Series GSE45146. Homo sapiens. 8 samples. Type: Non-coding RNA profiling by array.
Utility of CholangioFlex and Fluorescent in Situ Hybridization in the Diagnosis of Malignant Biliary Strictures
ClinicalTrials.gov study NCT01501474. IPD Sharing: Not stated. Countries: 1. Publications: 0.
GENSARC 2008 : Medical Economic Evaluation of the Molecular Detection by FISH (Fluorescent in Situ Hybridization) and by PCR (Polymerase Chain Reaction) of Sarcomas Specific Translocations and Amplifi
ClinicalTrials.gov study NCT00847691. IPD Sharing: Not stated. Countries: 1. Publications: 0.
Detection of Amplification of Her2 Fluorescent in Situ Hybridization (FISH) in Breast Tissue Sections
ClinicalTrials.gov study NCT01066507. IPD Sharing: Not stated. Countries: 2. Publications: 0.
Detection of Chromosomal Aberrations in Urine Samples Using Fluorescent in Situ Hybridization (FISH) (UroVysion Test)
ClinicalTrials.gov study NCT01264744. IPD Sharing: Not stated. Countries: 1. Publications: 0.
Detection of Chromosomal Aberrations in Non-small Cell Lung Cancer (NSCLC) Using Fluorescent in Situ Hybridization (FISH) (ALK Test)
ClinicalTrials.gov study NCT01620853. IPD Sharing: Not stated. Countries: 1. Publications: 0.
Disease Characteristics and Treatment Response in Plasma Cell Disorders Patients Based on Genetic Abnormalities From Fluorescence In Situ Hybridization and Next Generation Sequencing
ClinicalTrials.gov study NCT06330896. IPD Sharing: UNDECIDED. Countries: 0. Publications: 0.
Single-nucleus RNA sequencing combined with fluorescence in situ hybridization reveals Stat3 as a therapeutic target for ventilator-induced diaphragm dysfunction
GEO Series GSE276162. Mus musculus. 2 samples. Type: Expression profiling by high throughput sequencing.
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