Spatially Resolved Proteome Profiling of <200 Cells from Tomato Fruit Pericarp by Integrating Laser-Capture Microdissection with Nanodroplet Sample Preparation

2018 ◽  
Vol 90 (18) ◽  
pp. 11106-11114 ◽  
Author(s):  
Yiran Liang ◽  
Ying Zhu ◽  
Maowei Dou ◽  
Kerui Xu ◽  
Rosalie K. Chu ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Laser Capture Microdissection ◽  
Tomato Fruit ◽  
Proteome Profiling ◽  
Spatially Resolved ◽  
Fruit Pericarp ◽  
Laser Capture

scholarly journals Robust Acquisition of High Resolution Spatial Transcriptomes from Preserved Tissues with Immunofluorescence Based Laser Capture Microdissection

2021 ◽  
Author(s):  
Xiaodan Zhang ◽  
Chuansheng Hu ◽  
Chen Huang ◽  
Ying Wei ◽  
Xiaowei Li ◽  
...  
Keyword(s):  
Laser Capture Microdissection ◽  
Spatial Organization ◽  
Spatial Information ◽  
A Priori ◽  
Rna Degradation ◽  
Spatially Resolved ◽  
Mouse Small Intestine ◽  
Dissociated Cells ◽  
Laser Capture

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue. However, obtaining comprehensive transcriptomic data based on established phenotypes while retaining this spatial information has been challenging. Here we present a general and robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to enable acquisition of finely resolved spatial transcriptomes with as few as tens of cells from snap-frozen or RNAlater-treated tissues, overcoming the long-standing problem of significant RNA degradation during this lengthy process. The efficacy of this approach is exemplified by the characterization of differences at the transcript isoform level between cells at the tip versus the main capillary body of the mouse small intestine lacteal. With the extensive repertoire of phenotype-specific antibodies that are presently available, our method provides a powerful means by which spatially resolved cellular states can be delineated in situ with preserved tissues. Moreover, such high quality spatial transcriptomes defined by immuno-markers can be used to compare with clusters obtained from single-cell RNAseq studies of dissociated cells as well as applied to bead-based spatial transcriptomics approaches that require such information a priori for cell identification.

scholarly journals An optimised protocol for isolation of RNA through laser capture microdissection of leaf material

2019 ◽  
Author(s):  
Lei Hua ◽  
Julian M Hibberd
Keyword(s):  
Sample Preparation ◽  
Laser Capture Microdissection ◽  
Leaf Tissue ◽  
Cell Types ◽  
Rna Degradation ◽  
Mesophyll Cells ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Aqueous Conditions ◽  
Laser Capture

AbstractLaser Capture Microdissection is a powerful tool that allows thin slices of specific cells types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here we describe an optimized procedure to limit RNA degradation that is based on the use of low melting point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach high quality RNA suitable for down-stream applications such as quantitative reverse transcriptase polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.

scholarly journals Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

2019 ◽  
Author(s):  
Jeremy A. Herrera ◽  
Venkatesh Mallikarjun ◽  
Silvia Rosini ◽  
Maria Angeles Montero ◽  
Stacey Warwood ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Laser Capture Microdissection ◽  
Human Lung ◽  
Heat Extraction ◽  
Preparation Methods ◽  
Spatially Resolved ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Laser Capture ◽  
Formalin Fixed

ABSTRACTHaematoxylin and eosin (H&E) – which respectively stain nuclei blue and other cellular and stromal material pink – are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing, and H&E staining of tissues is poorly compatible with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique optimized to analyze H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. We advance our methodology by combining 3 techniques shown to individually enhance protein yields (heat extraction, physical disruption, and in column digestion) into one optimized pipeline for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE fixed section from Idiopathic Pulmonary Fibrosis (IPF) specimens were then subject to comparative proteomics using this methodology. This approach yielded 1252 differentially expressed proteins including 137 extracellular matrix (ECM) proteins. In addition, we offer proof of principal that MS can identify distinct, characteristic proteomic compositions of anatomical features within complex tissues.

A Novel Sample Preparation Method That Enables Nucleic Acid Analysis from Ultrathin Sections

2013 ◽  
Vol 19 (3) ◽  
pp. 635-641 ◽  
Author(s):  
Vincent P. Klink ◽  
Giselle Thibaudeau ◽  
Ronald Altig
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Laser Capture Microdissection ◽  
Preparation Method ◽  
Cell Types ◽  
Leopard Frog ◽  
Sample Preparation Method ◽  
Intact Cells ◽  
Nucleic Acid Analysis ◽  
Laser Capture

AbstractThe ability to isolate and perform nucleic acid analyses of individual cells is critical to studying the development of various cell types and structures. We present a novel biological sample preparation method developed for laser capture microdissection-assisted nucleic acid analysis of ultrathin cell/tissue sections. We used cells of the mitotic bed of the tadpole teeth of Lithobates sphenocephalus (Southern Leopard Frog). Cells from the mitotic beds at the base of the developing teeth series were isolated and embedded in the methacrylate resin, Technovit® 9100®. Intact cells of the mitotic beds were thin sectioned and examined by bright-field and transmission electron microscopy. The cytological and ultrastructural anatomy of the immature and progressively more mature tooth primordia appeared well preserved and intact. A developmental series of tooth primordia were isolated by laser capture microdissection (LCM). Processing of these cells for RNA showed that intact RNA could be isolated. The study demonstrates that Technovit® 9100® can be used as an embedding medium for extremely small tissues and from individual cells, a prerequisite step to LCM and nucleic acid analyses. A relatively small amount of sample material was needed for the analysis, which makes this technique ideal for cell-specific analyses when the desired cells are limited in quantity.

Laser microdissection and pressure-catapulting technique to study gene expression in the reoxygenated myocardium

2006 ◽  
Vol 290 (6) ◽  
pp. H2625-H2632 ◽  
Author(s):  
Donald E. Kuhn ◽  
Sashwati Roy ◽  
Jared Radtke ◽  
Sudip Gupta ◽  
Chandan K. Sen
Keyword(s):  
Myocardial Infarction ◽  
Laser Capture Microdissection ◽  
Laser Microdissection ◽  
Pcr Analysis ◽  
Staining Procedure ◽  
Infarct Core ◽  
Spatially Resolved ◽  
Biological Responses ◽  
Study Gene Expression ◽  
Laser Capture

For focal events such as myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with ∼80% loss in the 1st h. Tissue elements (1 × 104–4 × 106 μm2) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.

scholarly journals Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Jeremy A. Herrera ◽  
Venkatesh Mallikarjun ◽  
Silvia Rosini ◽  
Maria Angeles Montero ◽  
Craig Lawless ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Laser Capture Microdissection ◽  
Human Lung ◽  
Spatially Resolved ◽  
Laser Capture ◽  
Formalin Fixed
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