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Methods in Molecular Biology (Clifton,... 2024Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic...
Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.
Topics: Cryptococcus neoformans; Microdissection; Cellular Senescence; Lab-On-A-Chip Devices; Single-Cell Analysis; Cryptococcosis
PubMed: 38758331
DOI: 10.1007/978-1-0716-3722-7_25 -
Molecular Biotechnology Mar 2023An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory...
An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation.
Topics: Microdissection; RNA-Seq; Plants; Lasers; Quercus; RNA, Plant
PubMed: 35976558
DOI: 10.1007/s12033-022-00542-9 -
Journal of Microbiological Methods Jul 2017Laser microdissection is a method that allows for the isolation of homogenous cell populations from their native niches in tissues for downstream molecular assays. This... (Review)
Review
Laser microdissection is a method that allows for the isolation of homogenous cell populations from their native niches in tissues for downstream molecular assays. This method is widely used for genomic analysis, gene expression profiling and proteomic and metabolite assays in various fields of biology, but it remains an uncommon approach in microbiological research. In spite of the limited number of publications, laser microdissection was shown to be an extremely useful method for studying host-microorganism interactions in animals and plants, investigating bacteria within biofilms, identifying uncultivated bacteria and performing single prokaryotic cell analysis. The current paper describes the methodological aspects of commercially available laser microdissection instruments and representative examples that demonstrate the advantages of this method for resolving a variety of issues in microbiology.
Topics: Animals; Gene Expression Profiling; Host-Pathogen Interactions; Laser Capture Microdissection; Plants; Single-Cell Analysis
PubMed: 26775287
DOI: 10.1016/j.mimet.2016.01.001 -
Histology and Histopathology Nov 2015Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given... (Review)
Review
Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given tissue may be unique and cannot be homogenized across cell-types and spatial co-ordinates. For example, in response to myocardial infarction, on one hand myocytes and fibroblasts of the heart tissue respond differently. On the other hand, myocytes in the infarct core respond differently compared to those in the peri-infarct zone. Therefore, isolation of pure targeted cells is an important and essential step for the molecular analysis of cells involved in the progression of disease. Laser capture microdissection (LCM) is powerful to obtain a pure targeted cell subgroup, or even a single cell, quickly and precisely under the microscope, successfully tackling the problem of tissue heterogeneity in molecular analysis. This review presents an overview of LCM technology, the principles, advantages and limitations and its down-stream applications in the fields of proteomics, genomics and transcriptomics. With powerful technologies and appropriate applications, this technique provides unprecedented insights into cell biology from cells grown in their natural tissue habitat as opposed to those cultured in artificial petri dish conditions.
Topics: Animals; Biomarkers; Cell Separation; Gene Expression Profiling; Genetic Markers; Genomics; Humans; Laser Capture Microdissection; Proteomics
PubMed: 25892148
DOI: 10.14670/HH-11-622 -
Journal of Visualized Experiments : JoVE Mar 2022Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic "toolkits,"...
Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic "toolkits," such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis. LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species. Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.
Topics: Animals; Laser Capture Microdissection; Lasers; Male; Proteins; Transcriptome
PubMed: 35435919
DOI: 10.3791/63666 -
The Journal of Pathology Feb 2021The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially... (Comparative Study)
Comparative Study
The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Topics: Animals; Formaldehyde; Humans; Laser Capture Microdissection; Liver; Mice; Mice, Inbred C57BL; Neoplasms; Precision Medicine; Tissue Fixation
PubMed: 33135777
DOI: 10.1002/path.5575 -
Methods in Molecular Biology (Clifton,... 2016Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high...
Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high density molecular marker linkage map and fine physical map, the generation of probes for chromosome painting, and the localization and cloning of important genes. Here, we describe a modified technique to microdissect a single chromosome, paint individual chromosomes, and construct single-chromosome DNA libraries.
Topics: Chromosome Painting; Chromosomes, Plant; Cloning, Molecular; Gene Library; In Situ Hybridization, Fluorescence; Microdissection; Polymerase Chain Reaction; Secale
PubMed: 27511173
DOI: 10.1007/978-1-4939-3622-9_12 -
Journal of Visualized Experiments : JoVE Nov 2023Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF)....
Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy. We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.
Topics: Animals; Mice; Connexin 43; Pulmonary Veins; Microdissection; Myocardium; Atrial Fibrillation; Heart Atria; Fluorescent Antibody Technique; Catheter Ablation
PubMed: 38078615
DOI: 10.3791/65836 -
Omics : a Journal of Integrative Biology Sep 2014The concept of tissues appeared more than 200 years ago, since textures and attendant differences were described within the whole organism components. Instrumental... (Review)
Review
The concept of tissues appeared more than 200 years ago, since textures and attendant differences were described within the whole organism components. Instrumental developments in optics and biochemistry subsequently paved the way to transition from classical to molecular histology in order to decipher the molecular contexts associated with physiological or pathological development or function of a tissue. In 1941, Coons and colleagues performed the first systematic integrated examination of classical histology and biochemistry when his team localized pneumonia antigens in infected tissue sections. Most recently, in the early 21(st) century, mass spectrometry (MS) has progressively become one of the most valuable tools to analyze biomolecular compounds. Currently, sampling methods, biochemical procedures, and MS instrumentations allow scientists to perform "in depth" analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential "gold mines" for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between "classical" and "molecular" histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.
Topics: Histology; Immunohistochemistry; Laser Capture Microdissection; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 25105455
DOI: 10.1089/omi.2014.0033 -
Methods in Molecular Biology (Clifton,... 2021Testicular germ cell tumors are among the most common malignancies seen in children and young adults. Genomic studies have identified characteristic molecular profiles... (Review)
Review
Testicular germ cell tumors are among the most common malignancies seen in children and young adults. Genomic studies have identified characteristic molecular profiles in testicular cancer, which are associated with histologic subtypes and may predict clinical behavior including treatment responses. Emerging molecular technologies analyzing tumor genomics, transcriptomics, and proteomics may now guide precision management of testicular tumors. Laser-assisted microdissection methods such as laser capture microdissection efficiently isolate selected tumor cells from routine pathology specimens, avoiding contamination from nontarget cell populations. Laser capture microdissection in combination with next generation sequencing makes precise high throughput genetic evaluation effective and efficient. The use of laser capture microdissection (LCM) for molecular testing may translate into great benefits for the clinical management of patients with testicular cancers. This review discusses application protocols for laser-assisted microdissection to investigate testicular germ cell tumors.
Topics: Biomarkers, Tumor; Clinical Decision-Making; Diagnosis, Differential; Disease Management; Disease Susceptibility; Humans; Immunohistochemistry; Male; Microdissection; Molecular Diagnostic Techniques; Neoplasms, Germ Cell and Embryonal; Testicular Neoplasms
PubMed: 32852755
DOI: 10.1007/978-1-0716-0860-9_3