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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 -
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 -
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 -
Frontiers in Immunology 2023IgA nephropathy (IgAN), (LN), membranous nephropathy (MN), and minimal change nephropathy (MCN) are all belonged to autoimmune glomerulonephritis. This study aimed to...
BACKGROUND
IgA nephropathy (IgAN), (LN), membranous nephropathy (MN), and minimal change nephropathy (MCN) are all belonged to autoimmune glomerulonephritis. This study aimed to identify the specific proteomic characteristics of the four GNs diseases in order to provide frameworks for developing the appropriate drug for patients diagnosed with GNs disease.
METHODS
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized to investigate proteomic features of glomerular tissues obtained by laser capture microdissection (LCM). 8 normal control cases, 11 IgAN cases, 19 LN cases, 5 MN cases, and 3 MCN cases in this study were selected for bioinformatics analyses.
RESULTS
The shared overlapping proteins among the top 100 DEPs of each GNs type were mostly downregulated, in which only FLII was significantly downregulated in the four GNs diseases. A2M was significantly upregulated in MN, IgAN, and LN subgroups. The pathway of complement and coagulation cascades was notably activated with NES value ranging 2.77 to 3.39 among MCN, MN, IgAN, and LN diseases, but the pattern of protein expression level were significantly different. In LN patients, the increased activity of complement and coagulation cascades was contributed by the high expression of multiple complements (C1QB, C3, C4A, C4B, C6, C8B, C8G, C9). Meanwhile, both C1QC and C4B were remarkably upregulated in MN patients. On the contrary, complement-regulating proteins (CD59) was substantially decreased in MCN and IgAN subgroup.
CONCLUSIONS
The integrative proteomics analysis of the four GNs diseases provide insights into unique characteristics of GNs diseases and further serve as frameworks for precision medicine diagnosis and provide novel targets for drug development.
Topics: Humans; Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; Glomerulonephritis, IGA; Glomerulonephritis, Membranous; Nephrosis, Lipoid; Lasers
PubMed: 37033921
DOI: 10.3389/fimmu.2023.1131164 -
Annals of the New York Academy of... Nov 2020The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo... (Review)
Review
The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo models, progress to elucidate mechanisms of toxicity and to develop effective therapies, decontamination procedures, and general countermeasures is still limited. Novel scientific approaches to address these questions are needed to expand perspectives on existing knowledge and gain new insights. To achieve this, the use of ex vivo techniques like precision-cut tissue slices (PCTSs) can be a valuable approach. Existing studies employing this economical and relatively easy to implement method show model suitability and comparability with the use of in vitro and in vivo models. In this article, we review research on CWAs in PCTSs to illustrate the advantages of the approach and to promote future applications.
Topics: Animals; Chemical Warfare Agents; Humans; Microdissection
PubMed: 32808309
DOI: 10.1111/nyas.14459 -
International Journal of Molecular... Nov 2022The advancement in molecular techniques has been attributed to the quality and significance of cancer research. Pancreatic cancer (PC) is one of the rare cancers with... (Review)
Review
The advancement in molecular techniques has been attributed to the quality and significance of cancer research. Pancreatic cancer (PC) is one of the rare cancers with aggressive behavior and a high mortality rate. The asymptomatic nature of the disease until its advanced stage has resulted in late diagnosis as well as poor prognosis. The heterogeneous character of PC has complicated cancer development and progression studies. The analysis of bulk tissues of the disease was insufficient to understand the disease, hence, the introduction of the single-cell separating technique aided researchers to decipher more about the specific cell population of tumors. This review gives an overview of the Laser Capture Microdissection (LCM) technique, one of the single-cell separation methods used in PC research.
Topics: Humans; Laser Capture Microdissection; Pancreatic Neoplasms; Pancreas; Carcinoma, Pancreatic Ductal
PubMed: 36498893
DOI: 10.3390/ijms232314566 -
The Journal of Pathology Feb 2020Endosalpingiosis, a microscopic lesion composed of ectopic Fallopian tube epithelium, frequently involves the peritoneum and lymph nodes in patients with ovarian serous...
Endosalpingiosis, a microscopic lesion composed of ectopic Fallopian tube epithelium, frequently involves the peritoneum and lymph nodes in patients with ovarian serous borderline tumour or low-grade serous carcinoma, but its pathogenic significance remains unclear. Using laser-capture microdissection and droplet digital PCR, we investigated whether endosalpingiosis harbours the driver mutations in BRAF and KRAS that characterise ovarian low-grade serous neoplasms. Somatic mutations were detected in 14 (33%) of 43 endosalpingiotic lesions analysed. Of 21 women with endosalpingiosis associated with a synchronous or metachronous ovarian low-grade serous tumour, mutations were identified in endosalpingiotic lesions from 11 (52%) women, with most cases (10/11, 91%) demonstrating identical mutations in both tumour and endosalpingiosis. In contrast, of 13 cases of endosalpingiosis not associated with an ovarian tumour, only one harboured a KRAS mutation. The proliferative activity as assessed by Ki-67 immunohistochemistry was lower in endosalpingiosis than in low-grade serous tumours, and endosalpingiosis with either a BRAF or KRAS mutation had a significantly lower Ki-67 index than those without. Ectopic expression of KRAS in Fallopian tube epithelial cells led to ERK phosphorylation, p21 induction, growth arrest and cellular senescence. In conclusion, we demonstrate that endosalpingiosis represents an interesting example of cancer driver mutations in deceptively normal-appearing cells, which may be prone to neoplastic transformation upon bypass of endogenous oncosuppressive mechanisms. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Topics: Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Choristoma; Cystadenocarcinoma, Serous; Epithelial Cells; Fallopian Tubes; Female; Humans; Laser Capture Microdissection; Lymphatic Diseases; Mutation; Ovarian Neoplasms; Peritoneal Diseases; Precancerous Conditions; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras)
PubMed: 31576556
DOI: 10.1002/path.5353 -
Lab on a Chip Jul 2022Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most...
Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample amounts that blurs cell-type-specific or microstructure-specific information. In this study, we developed an improved sample preparation approach for spatial proteomics and integrated it with our previously-established laser capture microdissection (LCM) and microfluidics sample processing platform. Specifically, we developed a hanging drop (HD) method to improve the sample recovery by positioning a nanowell chip upside-down during protein extraction and tryptic digestion steps. Compared with the commonly-used sitting-drop method, the HD method keeps the tissue pixel away from the container surface, and thus improves the accessibility of the extraction/digestion buffer to the tissue sample. The HD method can increase the MS signal by 7 fold, leading to a 66% increase in the number of identified proteins. An average of 721, 1489, and 2521 proteins can be quantitatively profiled from laser-dissected 10 μm-thick mouse liver tissue pixels with areas of 0.0025, 0.01, and 0.04 mm, respectively. The improved system was further validated in the study of cell-type-specific proteomes of mouse uterine tissues.
Topics: Animals; Laser Capture Microdissection; Mice; Proteome; Proteomics; Specimen Handling; Workflow
PubMed: 35838077
DOI: 10.1039/d2lc00384h -
Cells Nov 2020Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran...
BACKGROUND
Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran families, structure of chromosome termini is usually conserved within genera. With the aim to assess whether or not the evolutionary distance between genera implies chromosome end diversification, this report exploits two representatives of Sciaridae, , and .
METHODS
Probes and plasmid microlibraries obtained by chromosome end microdissection, in situ hybridization, cloning, and sequencing are among the methodological approaches employed in this work.
RESULTS
The data argue for the existence of either specific terminal DNA sequences for each chromosome tip in , or sequences common to all chromosome ends but their extension does not allow detection by in situ hybridization. Both sciarid species share terminal sequences that are significantly underrepresented in chromosome ends of .
CONCLUSIONS
The data suggest an unusual terminal structure in chromosomes compared to other dipterans investigated. A putative, evolutionary process of repetitive DNA expansion that acted differentially to shape chromosome ends of the two flies is also discussed.
Topics: Animals; Base Sequence; Chromosomes, Insect; DNA; Diptera; Gene Library; Microdissection; Plasmids; Polytene Chromosomes
PubMed: 33167604
DOI: 10.3390/cells9112425