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An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

ABSTRACT: Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al.¹.

SUBMITTER: Jahun AS

PROVIDER: S-EPMC10901131 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

Jahun Aminu S AS Sorgeloos Frederic F Goodfellow Ian G IG

STAR protocols 20240222 1

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. ...[more]

PMID: 38393950

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Project description:The Cannabis market is experiencing steady global growth. Cannabis herbal extracts (CHE) are interesting and sought-after products for many clinical conditions. The medical potential of these formulations is mainly attributed to neutral cannabinoids, such as cannabidiol (CBD), tetrahydrocannabinol (THC), and cannabinol (CBN), and their non-standardized content poses a significant fragility in these pharmaceutical inputs. High-resolution mass spectrometry portrays a powerful alternative to their accurate monitoring; however, further analytical steps need to be critically optimized to keep up with instrumental performance. In this study, Full Factorial and Box-Behnken designs were employed to achieve a multivariate optimization of CBD, THC, and CBN extraction from human and veterinary commercial CHE using a minimum methanol/hexane 9:1 volume and short operational times. A predictive model was also constructed using the Response Surface Methodology and its accuracy was validated. Agitation and sonication times were identified as the most significant operational extraction parameters, followed by the extraction mixture volume. The final setup of the optimized method represented a significantly faster and cheaper protocol than those in the literature. The selected neutral cannabinoids quantification was conducted using ultra high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) with a precision of <15%, accuracy of 69-98%, sensitivity of 23-39 ng kg-1, and linearity regarding pharmaceutical requirements. State-of-the-art levels of analytical sensitivity and specificity were achieved in the target quantification due to high-resolution mass spectrometry. The developed method demonstrated reliability and feasibility for routine analytical applications. As a proof-of-concept, it enabled the efficient processing of 16 samples of commercial CHE within a three-hour timeframe, showcasing its practicality and reproducibility, and highlighting its potential for broader adoption in similar scenarios for both human and veterinary medicines.

Project description:IntroductionLow microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization.MethodsThree depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples.ResultsThe only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness.DiscussionDespite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

Dataset Information

An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

Publications

An optimized protocol for the extraction and quantification of cytosolic DNA in mammalian cells.

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