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.

Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies

PROTEOMICS ◽  
2011 ◽  
Vol 11 (17) ◽  
pp. 3556-3564 ◽  
Author(s):  
Wei Liu ◽  
Yufang Hou ◽  
Huahai Chen ◽  
Handong Wei ◽  
Weiran Lin ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Single Cell ◽  
Mouse Liver ◽  
Preparation Method ◽  
Cell Types ◽  
Sample Preparation Method

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 The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa

2020 ◽  
Author(s):  
Gert Johannes Kruger Marais ◽  
Michelle Naidoo ◽  
Nei-yuan Hsiao ◽  
Ziyaad Valley-Omar ◽  
Heidi Smuts ◽  
...  
Keyword(s):  
South Africa ◽  
Nucleic Acid ◽  
Sample Preparation ◽  
Preparation Method ◽  
Preparation Technique ◽  
Rt Pcr ◽  
Sample Preparation Method ◽  
Diagnostic Laboratory ◽  
Percent Agreement ◽  
Nucleic Acid Purification

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

A simple and innovative sample preparation method for on-site SARS-CoV-2 molecular diagnostics

The Analyst ◽  
2021 ◽  
Author(s):  
Songhyun LEE ◽  
Junkyu Song ◽  
Sanghyo Kim
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Molecular Diagnostics ◽  
Diagnostic Tool ◽  
Preparation Method ◽  
Nucleic Acid Amplification ◽  
Sample Preparation Method ◽  
Extraction Step

Nucleic acid amplification is a widely used diagnostic tool, although it requires a relatively time-consuming and complicated extraction step. To address this issue outside the laboratory, we investigated a sample...

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

Cross-Section Sample Preparation Method for Imaging Dopant Related Anomalies Using Scanning Probe Microscopy Techniques

2014 ◽  
Author(s):  
Swaminathan Subramanian ◽  
Khiem Ly ◽  
Tony Chrastecky
Keyword(s):  
Sample Preparation ◽  
Failure Analysis ◽  
Cross Section ◽  
Scanning Probe Microscopy ◽  
Preparation Method ◽  
Fault Isolation ◽  
Scanning Probe ◽  
Sample Preparation Method ◽  
Probe Microscopy ◽  
Section Sample

Abstract Visualization of dopant related anomalies in integrated circuits is extremely challenging. Cleaving of the die may not be possible in practical failure analysis situations that require extensive electrical fault isolation, where the failing die can be submitted of scanning probe microscopy analysis in various states such as partially depackaged die, backside thinned die, and so on. In advanced technologies, the circuit orientation in the wafer may not align with preferred crystallographic direction for cleaving the silicon or other substrates. In order to overcome these issues, a focused ion beam lift-out based approach for site-specific cross-section sample preparation is developed in this work. A directional mechanical polishing procedure to produce smooth damage-free surface for junction profiling is also implemented. Two failure analysis applications of the sample preparation method to visualize junction anomalies using scanning microwave microscopy are also discussed.

scholarly journals Application of the Dehydration Homogeneous Liquid–Liquid Extraction (DHLLE) Sample Preparation Method for Fingerprinting of Honey Volatiles

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2277
Author(s):  
Piotr M. Kuś ◽  
Igor Jerković
Keyword(s):  
Carboxylic Acid ◽  
Sample Preparation ◽  
Preparation Method ◽  
Liquid Extraction ◽  
Sample Preparation Method ◽  
Phenyllactic Acid ◽  
Homogeneous Liquid ◽  
Liquid Liquid Extraction ◽  
Wide Range ◽  
Chemical Groups

Recently, we proposed a new sample preparation method involving reduced solvent and sample usage, based on dehydration homogeneous liquid–liquid extraction (DHLLE) for the screening of volatiles and semi-volatiles from honey. In the present research, the method was applied to a wide range of honeys (21 different representative unifloral samples) to determine its suitability for detecting characteristic honey compounds from different chemical classes. GC-FID/MS disclosed 130 compounds from different structural and chemical groups. The DHLLE method allowed the extraction and identification of a wide range of previously reported specific and nonspecific marker compounds belonging to different chemical groups (including monoterpenes, norisoprenoids, benzene derivatives, or nitrogen compounds). For example, DHLLE allowed the detection of cornflower honey chemical markers: 3-oxo-retro-α-ionols, 3,4-dihydro-3-oxoedulan, phenyllactic acid; coffee honey markers: theobromine and caffeine; linden honey markers: 4-isopropenylcyclohexa-1,3-diene-1-carboxylic acid and 4-(2-hydroxy-2-propanyl)cyclohexa-1,3-diene-1-carboxylic acid, as well as furan derivatives from buckwheat honey. The obtained results were comparable with the previously reported data on markers of various honey varieties. Considering the application of much lower volumes of very common reagents, DHLLE may provide economical and ecological advantages as an alternative sample preparation method for routine purposes.

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