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42 results for “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.
Data for: Positive in situ hybridization for ZIKV RNA in MFI tissues and gravid uteri slides
<p>Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes, including severe birth defects and fetal/infant death. Potential pathways of vertical transmission <em>in utero</em> have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. The images included in this dataset belong to a study that aimed to determine the pathways of vertical transmission using a rhesus macaque model. Pregnant rhesus macaques were inoculated with 10<sup>4</sup> plaque forming using (PFA) of an African-lineage ZIKV isolate (ZIKV-DAK) at gestational day 30 (term is 165 days). Maternal-fetal interface and gravid uteri were evaluated for ZIKV RNA using <em>in situ</em> hybridization (ISH). The included images are the maternal fetal interface tissues and coronal sections evaluated for ZIKV via ISH, areas were ZIKV RNA was detected have pink/red staining. The pattern of infection observed in the maternal-fetal interface provides evidence of vertical ZIKV transmission through the fetal membranes.</p>
Data for the publication "In Situ Observation of Room-Temperature Magnesium Metal Deposition on a NASICON/IL Hybrid Solid Electrolyte"
<p>Datasets are obtained from X-ray diffraction (XRD), Rietveld analysis, Scanning electron microscopy (SEM), Electrochemicla impedance spectroscopy (EIS), Nudged Elastic Band (NEB), Chronopotentiometry (CP), Linear sweep voltammetry (LSV), X-ray photoelectron spectroscopy (XPS), Energy-dispersive X-ray spectroscopy (EDS).</p> <p><strong>Abstract:</strong> Secondary batteries using multivalent cations as ionic charge carriers have attracted increasing attention in recent years due to the high theoretical energy density provided by multi-electron redox reactions. However, the high charge density of these cations inevitably leads to sluggish kinetics of ion migration at room temperature, which poses a challenge for the development of solid-state batteries using multivalent ions. Here, we prepared a magnesium ion conducting hybrid solid electrolyte (HSE) consisting of a new NASICON-structured material, Mg0.5Sn2(PO4)3, and a small amount of Mg ionic liquid. The HSE shows superior room-temperature ionic conductivity of 1.11 · 10−4 S cm−1 and an activation energy of 0.36 eV. Due to good compatibility of the HSE with the magnesium metal anode, symmetric MgǀHSE|Mg cells show stable Mg plating and stripping behavior at room temperature. Using in situ electrochemical scanning electron microscopy measurements the room temperature growth-induced fracture of the HSE was observed, giving unequivocal evidence for magnesium deposition. These results may serve as starting point for understanding the magnesium deposition mechanism on solid electrolyte in solid-state batteries.</p>
Data for: Positive in situ hybridization for ZIKV RNA in MFI tissues and gravid uteri slides
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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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Data from: Distinguishing the victim from the threat: SNP‐based methods reveal the extent of introgressive hybridization between wildcats and domestic cats in Scotland and inform future in situ and ex situ management options for species restoration
The degree of introgressive hybridisation between the Scottish wildcat and domestic cat has long been suspected to be advanced. Here we use a 35-SNP-marker test, designed to assess hybridisation between wildcat and domestic cat populations in Scotland, to assess a database of 265 wild-living and captive cat samples, and test the assumptions of the test using 3097 SNP markers generated independently in a subset of the data using ddRAD. We discovered that despite increased genetic resolution provided by these methods, wild-living cats in Scotland show a complete genetic continuum or hybrid swarm structure when judged against reference data. The historical population of wildcats, although hybridised, clearly groups at one end of this continuum, as does the captive population of wildcats. The interpretation of pelage scores against nuclear genetic data continues to be problematic. This is probably because of a breakdown in linkage disequilibrium between wildcat pelage genes as the two populations have become increasingly mixed, meaning that pelage score or SNP score alone are poor diagnostic predictors of hybrid status. Until better tools become available, both should be used jointly, where possible, when making management decisions about individual cats. We recommend that the conservation community in Scotland must now define clearly what measures are to be used to diagnose a wildcat in the wild in Scotland, if future conservation action is to be effective.
Data from: Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation
Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value.
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>
ZIKV in situ hybridization from full thickness placental center cuts, only positive samples included
<p>These data accompany the manuscript "Infection of the maternal-fetal interface and vertical transmission following low-dose inoculation of pregnant rhesus macaques (<em>Macaca mulattta</em>) with an African-lineage Zika virus" The included images are low magnification scans of full-thickness placental sections (placenta containing decidua, placental villi, and chorionic plate) evaluated with in situ hybridization against ZIKV RNA. Details of methods are provided in the manuscript. Only slides that were deemed positive are included. </p>
In situ tuning of dynamical Coulomb blockade in hybrid nanowire devices
<p>This repository contains the raw data and processing Python scripts corresponding to the paper "In situ tuning of dynamical Coulomb blockade in hybrid nanowire devices"</p>
Data from: Distinguishing the victim from the threat: SNP‐based methods reveal the extent of introgressive hybridization between wildcats and domestic cats in Scotland and inform future in situ and ex situ management options for species restoration
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ZIKV in situ hybridization from full thickness placental center cuts, only positive samples included
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Data from: Evolution in situ: hybrid origin and establishment of willows (Salix L.) on alpine glacier forefields
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Data from: Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation
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Data from: Molecular genetics unveiled unknown family relationships and hybrids in an ex-situ colony of African penguins (Spheniscus demersus)
Genealogical relationships among colony members, inbreeding status, and presence of hybrids are crucial data that can assist zoo curators in captive colony management and decision-making on relocation for reproduction. This study employed molecular markers to study a large colony (n=56) of African Penguin hosted in an Italian biopark. A panel of 15 STRs (single tandem repeats) was selected, and genotype data were analyzed using COLONY software to determine parentage relationships and compare the existing studbook information to a pedigree built from genetic analyses. The existence of extra-pair mating and the presence of hybrids were investigated: discrepancies in kinship relationships emerged following molecular parentage analysis and ten unknown genetic relationships were revealed. Infidelity of one member of the pair was observed in six cases and extra-pair copulation was assessed by genetic analysis in two episodes. One member of the colony was found to be a hybrid (S. demersus X S. humboldti); his progeny, derived by extra-pair copulation, was traced. Three other hidden hybrids were discovered and assessed using the identified candidate private alleles. Overall, our results demonstrate that molecular methods to confirm parentage and analyze relatedness among colony members are a valuable tool to complement studbook-based genetic management of African penguin captive populations. Because a variety of behavioral dynamics (e.g., extra-pair mating) can make observations ineffective in some species and because molecular markers outperform studbook in identifying the presence of hybrids, reliance on studbook information alone is not recommended.
In situ hybridization probe sequences for mouse Alx3, Fgf8, Msx1, Shh, Six3
<p><span><span><span><span><span><span><span><span><span><span><span>Serum response factor (SRF) is an essential transcription factor that influences many cellular processes including cell proliferation, migration, and differentiation. SRF directly regulates and is required for immediate early gene (IEG) and actin cytoskeleton-related gene expression. SRF coordinates these competing transcription programs through discrete sets of cofactors, the Ternary Complex Factors (TCFs) and Myocardin Related Transcription Factors (MRTFs). The relative contribution of these two programs to <i>in vivo</i> SRF activity and mutant phenotypes is not fully understood. To study how SRF utilizes its cofactors during development, we generated a knock-in <i>Srf</i><i><sup>a</sup></i><i><sup>I</sup></i> allele in mice harboring point mutations that disrupt SRF-MRTF-DNA complex formation but leave SRF-TCF activity unaffected. Homozygous <i>Srf</i><i><sup>a</sup></i><i><sup>I/</sup></i><i><sup>a</sup></i><i><sup>I</sup></i> mutants die at E10.5 with notable cardiovascular phenotypes, and neural crest conditional mutants succumb at birth to defects of the cardiac outflow tract but display none of the craniofacial phenotypes associated with complete loss of SRF in that lineage. Our studies further support an important role for MRTF mediating SRF function in cardiac neural crest and suggest new mechanisms by which SRF regulates transcription during development.</span></span></span></span></span></span></span></span></span></span></span></p>
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Allen Brain Atlas
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