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The impact of ultraviolet- and infrared-based laser microdissection technology on phosphoprotein detection in the laser microdissection-reverse phase protein array workflow
摘要： Reversible protein phosphorylation represents a key mechanism by which signals are transduced in eukaryotic cells. Dysregulated phosphorylation is also a hallmark of carcinogenesis and represents key drug targets in the precision medicine space. Thus, methods that preserve phosphoprotein integrity in the context of clinical tissue analyses are crucially important in cancer research. Here we investigated the impact of UV laser microdissection (UV LMD) and IR laser capture microdissection (IR LCM) on phosphoprotein abundance of key cancer signaling protein targets assessed by reverse-phase protein microarray (RPPA). Tumor epithelial cells from consecutive thin sections obtained from four high-grade serous ovarian cancers were harvested using either UV LMD or IR LCM methods. Phosphoprotein abundances for ten phosphoproteins that represent important drug targets were assessed by RPPA and revealed no significant differences in phosphoprotein integrity from those obtained using higher-energy UV versus the lower-energy IR laser methods.
关键词： Reverse phase protein array,Phosphoprotein,Proteomics,Laser capture microdissection,Laser microdissection
更新于2025-09-19 17:13:59
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Combining laser microdissection and microRNA expression profiling to unmask microRNA signatures in complex tissues
摘要： Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression information from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment-specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopathology. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates.
关键词： laser microdissection (LMD),biomarker discovery,tumor microenvironment,drug discovery,microRNA profiling,tissue complexity
更新于2025-09-19 17:13:59
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Laser microdissection-based microproteomics of the hippocampus of a rat epilepsy model reveals regional differences in protein abundances
摘要： Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder affecting almost 40% of adult patients with epilepsy. Hippocampal sclerosis (HS) is a common histopathological abnormality found in patients with MTLE. HS is characterised by extensive neuronal loss in different hippocampus sub-regions. In this study, we used laser microdissection-based microproteomics to determine the protein abundances in different regions and layers of the hippocampus dentate gyrus (DG) in an electric stimulation rodent model which displays classical HS damage similar to that found in patients with MTLE. Our results indicate that there are differences in the proteomic profiles of different layers (granule cell and molecular), as well as different regions, of the DG (ventral and dorsal). We have identified new signalling pathways and proteins present in specific layers and regions of the DG, such as PARK7, RACK1, and connexin 31/gap junction. We also found two major signalling pathways that are common to all layers and regions: inflammation and energy metabolism. Finally, our results highlight the utility of high-throughput microproteomics and spatial-limited isolation of tissues in the study of complex disorders to fully appreciate the large biological heterogeneity present in different cell populations within the central nervous system.
关键词： microproteomics,rat epilepsy model,hippocampus,protein abundances,Laser microdissection,PARK7,energy metabolism,connexin 31/gap junction,RACK1,inflammation
更新于2025-09-19 17:13:59
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Proteomics of human glomerulonephritis by laser microdissection and liquid chromatography‐tandem mass spectrometry
摘要： Aim: Laser microdissection (LMD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) enable clinicians to analyse proteins from tissue sections. In nephrology, these methods are used to diagnose diseases of abnormal protein deposition, such as amyloidosis, but they are seldom applied to the diagnosis and pathophysiological understanding of human glomerular diseases. Methods: Renal biopsy specimens were obtained from five patients with IgA nephropathy (IgAN), five patients with membranous nephropathy (MN) and five kidney transplant donors (as controls). From 10-μm-thick sections of formalin-fixed, paraffin-embedded specimens, 0.3-mm2 samples of glomerular tissue were subjected to LMD. The samples were analysed by LC-MS/MS and investigated clinically and histologically. Results: From the control glomeruli, we identified more than 300 types of proteins. In patients with IgAN, we detected significant increases not only in IgA1 and in C3 but also in the factors related to oxidative stress and cell proliferation in comparison to the controls. In patients with MN, levels of IgG1, IgG4, C3, C4a, and phospholipase-A2-receptor were significantly elevated in comparison to the controls, as were the aforementioned factors related to oxidative stress and cell proliferations detected in IgAN. Conclusion: Application of LMD and LC-MS/MS to renal biopsy specimens enabled us to identify not only pathognomonic proteins for the diagnosis but also several factors possibly involved in the pathogenesis of human glomerular diseases.
关键词： glomerulonephritis,IgA nephropathy,membranous nephropathy,laser microdissection,mass spectrometry
更新于2025-09-12 10:27:22
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A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms
摘要： Background: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. Results: A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. Conclusion: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes.
关键词： Non-model species,Cryosectioning,Evo-devo,Development,RNA-seq,Laser microdissection (LMD)
更新于2025-09-12 10:27:22
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Laser microdissection system applied in improving FT-TIMS technique
摘要： A laser microdissection system was used for isolating fission track stars to improve the cutting efficiency in the FT-TIMS particle analytical technique. The background interference of track detectors to determine isotopic ratios was studied. Several swipe samples were successfully measured for particle analysis by FT-TIMS. The results show that the laser microdissection system could effectively improve the ability of FT-TIMS particle analytical technique.
关键词： Isotopic ratios,Particle analysis,Laser microdissection,Fission tracks
更新于2025-09-11 14:15:04
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Concordance of KRAS mutation status between luminal and peripheral regions of primary colorectal cancer. A laser-capture microdissection-based study
摘要： The presence of KRAS mutation in colorectal cancer (CRC) is a marker of resistance to anti-EGFR therapy. However, there are conflicting reports concerning intratumoral heterogeneity of KRAS mutations. The aim of this study was to determine whether within primary CRCs with KRAS mutations intratumoral KRAS mutation heterogeneity can be detected between two strictly defined areas, i.e. the luminal (mucosa/submucosa) and peripheral invasive front of the tumor. Using laser-capture microdissection, from every tumor about 400-500 nests of cancer cells were excised from each of the examined areas (luminal and peripheral) and PNAClamp, a high-sensitivity real-time PCR-based diagnostic assay for KRAS mutation testing, was used for molecular analysis. KRAS mutations were detected in codon 12 in both luminal and peripheral regions in all tumors examined. We conclude that from the point of view of practical KRAS mutation testing for predictive purposes in patients with CRC (i.e. testing mutations in codons 12 and 13) sampling errors are unlikely to occur if in CRCs with KRAS mutations only the luminal (as in biopsy tissue) or peripheral region is examined, provided a sensitive system of detection is applied and an appropriate number of tumor cells with minimal contamination by benign cells is analyzed.
关键词： laser microdissection,KRAS mutation,colorectal cancer,KRAS intratumoral homogeneity
更新于2025-09-11 14:15:04