Optimization of microbial DNA extraction from human skin samples for CRISPR typing

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

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Standard Preparation Protocol of Human Skin Samples for Transmission Electron Microscopy

Skin Tissue Engineering - Methods in Molecular Biology ◽

10.1007/978-1-4939-9473-1_16 ◽

2019 ◽

pp. 205-215

Author(s):

Gery Barmettler ◽

Urs Ziegler

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Human Skin ◽

Standard Preparation ◽

Transmission Electron ◽

Skin Samples

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Capturing Age-Dependent Properties of Human Skin Using Magnetorheological Elastomers

Volume 1: Development and Characterization of Multifunctional Materials; Modeling, Simulation, and Control of Adaptive Systems; Integrated System Design and Implementation ◽

10.1115/smasis2018-8015 ◽

2018 ◽

Author(s):

Kyle Weaver ◽

Jeong-Hoi Koo ◽

Tae-Heon Yang ◽

Young-Min Kim

Keyword(s):

Mechanical Properties ◽

Human Skin ◽

Skin Testing ◽

Filler Particle ◽

Variable Stiffness ◽

Particle Volume ◽

Magnetorheological Elastomers ◽

Age Dependent ◽

Skin Samples

Artificial and synthetic skins are widely used in the medical field; used in applications ranging from skin grafts to suture training pads. There is a growing need for artificial skins with tunable properties. However, current artificial skins do not take into account the variability of mechanical properties between individual humans as well as the age-dependent properties of human skin. Furthermore, there has been little development in artificial skins based on these properties. Thus, the primary purpose of this research is to develop variable stiffness artificial skin samples using magnetorheological elastomers (MREs) whose properties that can be controlled using external magnetic fields. In this study, multiple MRE skin samples were fabricated with varying filler particle volume contents. Using a precision dynamic mechanical analyzer, a series of indenting experiments were performed on the samples to characterize their mechanical properties. The samples were tested using a spherical indenter that indented a total depth of 1 mm with a speed of 0.01 mm/s and unloaded at the same rate. The results show that the modulus or stiffness increases significantly as the iron percent (w/w) in the sample increases. Additionally, the stiffness of the sample increases proportional to the intensity of the applied external magnetic field. To assess the MRE samples’ variability of properties, the testing results were compared with in vivo human skin testing data. The results show the MRE samples are feasible to represent the age-dependent stiffness demonstrated in in vivo human skin testing. The MRE materials studied will be further studied as a variable-stiffness skin model in medical devices, such as radial pulse simulators.

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Gas Chromatographic Determination of Amino Acids and Polyamines in Human Skin Samples using Trifluoroacetylacetone and Isobutyl Chloroformate as Derivatizing Reagents

Journal of Chromatography & Separation Techniques ◽

10.4172/2157-7064.1000248 ◽

2014 ◽

Vol 05 (06) ◽

Cited By ~ 1

Author(s):

Sohail Ahmed Soomro

Keyword(s):

Amino Acids ◽

Human Skin ◽

Chromatographic Determination ◽

Isobutyl Chloroformate ◽

Skin Samples

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Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

10.1101/2021.10.19.464972 ◽

2021 ◽

Author(s):

Shunsuke Matsuoka ◽

Yoriko Sugiyama ◽

Mariko Nagano ◽

Hideyuki Doi

Keyword(s):

Dna Extraction ◽

Environmental Dna ◽

Operational Taxonomic Unit ◽

Pcr Inhibition ◽

Dna Metabarcoding ◽

Fungal Dna ◽

Diversity Studies ◽

Microbial Dna ◽

Methodological Studies ◽

Biodiversity Analysis

Background: Environmental DNA (eDNA) metabarcoding is a rapidly expanding technique for efficient biodiversity monitoring, especially of animals. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of the DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results [i.e., operational taxonomic unit (OTU) number and composition] of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

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