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.

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.

scholarly journals Laser Capture Microdissection in Dentistry

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Uraiwan Chokechanachaisakul ◽  
Tomoatsu Kaneko ◽  
Takashi Okiji ◽  
Reika Kaneko ◽  
Hideaki Suda ◽  
...  
Keyword(s):  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Cell Types ◽  
Specific Cell ◽  
Tissue Sections ◽  
Rna Analysis ◽  
Molecular Biological Analysis ◽  
Polymerase Chain ◽  
Tissue Specimens ◽  
Laser Capture

Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry.

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 Salinity tolerance in chickpea is associated with the ability to ‘exclude’ Na from leaf mesophyll cells

2019 ◽  
Vol 70 (18) ◽  
pp. 4991-5002 ◽  
Author(s):  
Lukasz Kotula ◽  
Peta L Clode ◽  
Juan De La Cruz Jimenez ◽  
Timothy D Colmer
Keyword(s):  
Salinity Tolerance ◽  
Structural Damage ◽  
Leaf Tissue ◽  
Cell Types ◽  
Epidermal Cells ◽  
Salinity Treatment ◽  
Mesophyll Cells ◽  
X Ray ◽  
Leaf Mesophyll ◽  
Excessive Accumulation

Abstract Salinity tolerance is associated with Na ‘exclusion’ from, or ‘tissue tolerance’ in, leaves. We investigated whether two contrasting chickpea genotypes, salt-tolerant Genesis836 and salt-sensitive Rupali, differ in leaf tissue tolerance to NaCl. We used X-ray microanalysis to evaluate cellular Na, Cl, and K concentrations in various cell types within leaflets and also in secretory trichomes of the two chickpea genotypes in relation to photosynthesis in control and saline conditions. TEM was used to assess the effects of salinity on the ultrastructure of chloroplasts. Genesis836 maintained net photosynthetic rates (A) for the 21 d of salinity treatment (60 mM NaCl), whereas A in Rupali substantially decreased after 11 d. Leaflet tissue [Na] was low in Genesis836 but had increased markedly in Rupali. In Genesis836, Na was accumulated in epidermal cells but was low in mesophyll cells, whereas in Rupali cellular [Na] was high in both cell types. The excessive accumulation of Na in mesophyll cells of Rupali corresponded to structural damage to the chloroplasts. Maintenance of photosynthesis and thus salinity tolerance in Genesis836 was associated with an ability to ‘exclude’ Na from leaflets and in particular from the photosynthetically active mesophyll cells, and to compartmentalize Na in epidermal cells.

scholarly journals Analysis of Genome-Wide Monoallelic Expression Patterns in Three Major Cell Types of Mouse Visual Cortex Using Laser Capture Microdissection

PLoS ONE ◽  
2016 ◽  
Vol 11 (9) ◽  
pp. e0163663 ◽  
Author(s):  
Chia-Yi Lin ◽  
Shih-Chuan Huang ◽  
Chun-Che Tung ◽  
Chih-Hsuan Chou ◽  
Susan Shur-Fen Gau ◽  
...  
Keyword(s):  
Visual Cortex ◽  
Laser Capture Microdissection ◽  
Expression Patterns ◽  
Cell Types ◽  
Monoallelic Expression ◽  
Genome Wide ◽  
Mouse Visual Cortex ◽  
Laser Capture

Reproducibility of Protein Identification of Selected Cell Types in Barrett’s Esophagus Analyzed by Combining Laser-Capture Microdissection and Mass Spectrometry

2011 ◽  
Vol 10 (1) ◽  
pp. 288-298 ◽  
Author(s):  
Christoph Stingl ◽  
Frederike G. I. van Vilsteren ◽  
Coskun Guzel ◽  
Fiebo J. W. ten Kate ◽  
Mike Visser ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Barrett’S Esophagus ◽  
Laser Capture Microdissection ◽  
Barrett's Esophagus ◽  
Protein Identification ◽  
Cell Types ◽  
Laser Capture

scholarly journals Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection

2004 ◽  
Vol 181 (1) ◽  
pp. 157-167 ◽  
Author(s):  
S Ho Hong ◽  
H Young Nah ◽  
J Yoon Lee ◽  
M Chan Gye ◽  
C Hoon Kim ◽  
...  
Keyword(s):  
Cdna Microarray ◽  
Laser Capture Microdissection ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Cell Types ◽  
Integral Membrane Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mouse Uterus ◽  
Laser Capture

The steroid hormone, estrogen, plays an important role in various physiological events which are mediated via its nuclear estrogen receptors, ERalpha and ERbeta. However, the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. To identify genes that are regulated by estrogen, the ovariectomized mouse uterus was exposed to 17beta-estradiol (E2) for 6 h and 12 h, and the data were analyzed by cDNA microarray. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include small proline-rich protein 2A, receptor-activity-modifying protein 3, inhibitor of DNA binding-1, eukaryotic translation initiation factor 2, cystatin B, decorin, secreted frizzled-related protein 2, integral membrane protein 2B and chemokine ligand 12. The expression patterns of several selected genes identified by the microarray analysis were confirmed by RT-PCR. In addition, laser capture microdissection (LCM) was conducted to determine the expression of selected genes in specific uterine cell types. Analysis of early and late responsive genes using LCM and cDNA microarray not only suggests direct and indirect effects of E2 on uterine physiological events, but also demonstrates differential regulation of E2 in specific uterine cell types. These results provide a basic background on global gene alterations or genetic pathways in the uterus during the estrous cycle and the implantation period.

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