Comparison of extraction methods for intracellular metabolomics

2021 ◽  
Author(s):  
Carolin Andresen ◽  
Tobias Boch ◽  
Hagen M. Gegner ◽  
Nils Mechtel ◽  
Andreas Narr ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Extraction Methods ◽  
Biological Information ◽  
Global Estimate ◽  
Regulatory Processes ◽  
Online Resource ◽  
Human Sample ◽  
Shiny App ◽  
R Shiny ◽  
Comprehensive Study

Measurements of metabolic compounds inside cells or tissues are of high informative potential since they represent the endpoint of biological information flow and a snapshot of the integration of many regulatory processes. However, it requires careful extraction to quantify their abundance. Here we present a comprehensive study using ten extraction protocols on four human sample types (liver tissue, bone marrow, HL60 and HEK cells) targeting 630 metabolites of different chemical classes. We show that the extraction efficiency and stability is highly variable across protocols and tissues by using different quality metrics including the limit of detection and variability between replicates as well as the sum of concentration as a global estimate of extraction stability. The profile of extracted metabolites depends on the used solvents - an observation which has implications for measurements of different sample types and metabolic compounds of interest. To identify the optimal extraction method for future metabolomics studies, the benchmark dataset was implemented in an easy-to-use, interactive and flexible online resource (R/shiny app MetaboExtract).

Unified exact design with early stopping rules for single arm clinical trials with multiple endpoints

2021 ◽  
pp. 096228022110130
Author(s):  
Wei Wei ◽  
Denise Esserman ◽  
Michael Kane ◽  
Daniel Zelterman
Keyword(s):  
Clinical Trials ◽  
Early Phase ◽  
Decision Rules ◽  
Stopping Rules ◽  
Multiple Endpoints ◽  
Shiny App ◽  
R Shiny ◽  
Early Phase Clinical Trials ◽  
User Friendly ◽  
Exact Design

Adaptive designs are gaining popularity in early phase clinical trials because they enable investigators to change the course of a study in response to accumulating data. We propose a novel design to simultaneously monitor several endpoints. These include efficacy, futility, toxicity and other outcomes in early phase, single-arm studies. We construct a recursive relationship to compute the exact probabilities of stopping for any combination of endpoints without the need for simulation, given pre-specified decision rules. The proposed design is flexible in the number and timing of interim analyses. A R Shiny app with user-friendly web interface has been created to facilitate the implementation of the proposed design.

scholarly journals Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 240
Author(s):  
Alison Woodward ◽  
Alina Pandele ◽  
Salah Abdelrazig ◽  
Catherine A. Ortori ◽  
Iqbal Khan ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Extraction Method ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Serum Starvation ◽  
Extraction Techniques ◽  
Metabolic Genes ◽  
Dual Extraction

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

scholarly journals Determination of Trace Antimony (III) in Water Samples with Single Drop Microextraction Using BPHA-[C4mim][PF6] System Followed by Graphite Furnace Atomic Absorption Spectrometry

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaoshan Huang ◽  
Mingxin Guan ◽  
Zhuliangzi Lu ◽  
Yiping Hang
Keyword(s):  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Limit Of Detection ◽  
Graphite Furnace ◽  
Absorption Spectrometry ◽  
Extraction Methods ◽  
Single Drop ◽  
Single Drop Microextraction ◽  
Relative Standard ◽  
Graphite Furnace Atomic Absorption

A new sensitive method for antimony (III) determination by graphite furnace atomic absorption spectrometry (GFAAS) has been developed by using N-benzoyl-N-phenylhydroxylamine (BPHA) and 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6]) single drop microextraction. The single drop microextraction (SDMM) system is more competitive compared with other traditional extraction methods. Under the optimized conditions, the limit of detection (signal-to-noise ratio is 3) and the enrichment factor of antimony (III) are 0.01 μg·L−1 and 112, respectively. The relative standard deviation of the 0.5 μg·L−1 antimony (III) is 4.2% (n=6). The proposed method is rather sensitive to determinate trace antimony (III) in water.

Effect of Extraction Systems on Clomazone Recovery from Aged Soil Samples

1995 ◽  
Vol 78 (6) ◽  
pp. 1519-1521 ◽  
Author(s):  
K Bruce Kirksey ◽  
Thomas C Mueller
Keyword(s):  
Liquid Chromatography ◽  
Limit Of Detection ◽  
Extraction Methods ◽  
Reversed Phase ◽  
Soil Samples ◽  
Moist Soil ◽  
Reversed Phase Liquid Chromatography ◽  
Extraction Solvent ◽  
Rotary Evaporator ◽  
Phase Liquid

Abstract Seven extraction methods were examined to determine which one provided a simple, efficient clomazone extraction from aged soil. The method selected involved adding 80 mL acetonitrile to moist soil and shaking for 16 h. The extract was filtered, and an additional 80 mL acetonitrile was added, equilibrated for 1 h, and filtered. Filtrates were combined and concentrated with a rotary evaporator. Clomazone concentrations were then determined by reversed-phase liquid chromatography with UV detection at 220 nm. Recovery with this method was equal to a 16 h + 1 h extraction with methanol, but acetonitrile was selected as extraction solvent because of its ease of removal with a rotary evaporator. The recovery from fortified soil samples was >80%, and a conservative lower limit of detection was 40 ng/g soil.

scholarly journals Transcriptional Differences for COVID-19 Disease Map Genes between Males and Females Indicate a Different Basal Immunophenotype Relevant to the Disease

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1447
Author(s):  
Tianyuan Liu ◽  
Leandro Balzano-Nogueira ◽  
Ana Lleo ◽  
Ana Conesa
Keyword(s):  
Gene Expression ◽  
Disease Incidence ◽  
Tissue Expression ◽  
Matrix Metalloproteases ◽  
Sex And Gender ◽  
Demographic Groups ◽  
Shiny App ◽  
R Shiny ◽  
And Gender ◽  
High Level

Worldwide COVID-19 epidemiology data indicate differences in disease incidence amongst sex and gender demographic groups. Specifically, male patients are at a higher death risk than female patients, and the older population is significantly more affected than young individuals. Whether this difference is a consequence of a pre-existing differential response to the virus, has not been studied in detail. We created DeCovid, an R shiny app that combines gene expression (GE) data of different human tissue from the Genotype-Tissue Expression (GTEx) project along with the COVID-19 Disease Map and COVID-19 related pathways gene collections to explore basal GE differences across healthy demographic groups. We used this app to study differential gene expression of COVID-19 associated genes in different age and sex groups. We identified that healthy women show higher expression-levels of interferon genes. Conversely, healthy men exhibit higher levels of proinflammatory cytokines. Additionally, young people present a stronger complement system and maintain a high level of matrix metalloproteases than older adults. Our data suggest the existence of different basal immunophenotypes amongst different demographic groups, which are relevant to COVID-19 progression and may contribute to explaining sex and age biases in disease severity. The DeCovid app is an effective and easy to use tool for exploring the GE levels relevant to COVID-19 across demographic groups and tissues.

scholarly journals Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

Open Medicine ◽  
2008 ◽  
Vol 3 (2) ◽  
pp. 157-162
Author(s):  
Koray Ergunay ◽  
Pinar Yurdakul ◽  
Burcin Sener ◽  
Ugur Ozcelik ◽  
Erdem Karabulut ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Burkholderia Cepacia ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Burkholderia Cepacia Complex ◽  
Assay Sensitivity ◽  
Spin Column ◽  
Significant Difference

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

scholarly journals Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247851
Author(s):  
Amy Oreskovic ◽  
Barry R. Lutz
Keyword(s):  
Large Volume ◽  
Magnetic Beads ◽  
Limit Of Detection ◽  
Extraction Methods ◽  
Urine Samples ◽  
Hybridization Method ◽  
Analytical Methodology ◽  
Cell Free Dna ◽  
Double Stranded Dna ◽  
Free Dna

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in Food

2005 ◽  
Vol 68 (6) ◽  
pp. 1294-1301 ◽  
Author(s):  
ERIC A. E. GARBER ◽  
ROBERT M. EPPLEY ◽  
MICHAEL E. STACK ◽  
MICHAEL A. McLAUGHLIN ◽  
DOUGLAS L. PARK
Keyword(s):  
High Throughput Screening ◽  
Limit Of Detection ◽  
Extraction Methods ◽  
Food Samples ◽  
Lateral Flow ◽  
Health Concern ◽  
High Background ◽  
Baked Goods ◽  
Simple Extraction ◽  
Flow Devices

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values ≥0.1 mg/kg of body weight. Ricin (oral LD50 &gt; 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 &gt; 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically ≤0.020 μg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

scholarly journals methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data

2020 ◽  
Author(s):  
Parker Knight ◽  
Marie-Pierre L. Gauthier ◽  
Carolina E. Pardo ◽  
Russell P. Darst ◽  
Alberto Riva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Single Molecule ◽  
Nucleosome Occupancy ◽  
Chromatin Accessibility ◽  
Biologically Relevant ◽  
Shiny App ◽  
R Shiny ◽  
Long Read ◽  
Cell Data

AbstractDifferential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we developed methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin landscapes. Methylscaper implements a weighted principle component analysis that orders sequencing reads, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper’s utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets.Availability and implementationMethylscaper, is available on GitHub at https://github.com/rhondabacher/[email protected]

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