Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
- Publikationstyp:
- Zeitschriftenaufsatz
- Metadaten:
-
- Autoren
- Elmo WI Neuberger
- Alexandra Brahmer
- Tobias Ehlert
- Katrin Kluge
- Keito FA Philippi
- Simone C Boedecker
- Julia Weinmann-Menke
- Perikles Simon
- Autoren-URL
- https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000671791200010&DestLinkType=FullRecord&DestApp=WOS_CPL
- DOI
- 10.1038/s41598-021-92826-4
- Externe Identifier
- Clarivate Analytics Document Solution ID: TH0NI
- PubMed Identifier: 34193884
- ISSN
- 2045-2322
- Ausgabe der Veröffentlichung
- 1
- Zeitschrift
- SCIENTIFIC REPORTS
- Artikelnummer
- ARTN 13581
- Datum der Veröffentlichung
- 2021
- Status
- Published
- Titel
- Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
- Sub types
- Article
- Ausgabe der Zeitschrift
- 11
Datenquelle: Web of Science (Lite)
- Andere Metadatenquellen:
-
- Abstract
- <jats:title>Abstract</jats:title><jats:p>Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.</jats:p>
- Autoren
- Elmo WI Neuberger
- Alexandra Brahmer
- Tobias Ehlert
- Katrin Kluge
- Keito FA Philippi
- Simone C Boedecker
- Julia Weinmann-Menke
- Perikles Simon
- DOI
- 10.1038/s41598-021-92826-4
- eISSN
- 2045-2322
- Ausgabe der Veröffentlichung
- 1
- Zeitschrift
- Scientific Reports
- Sprache
- en
- Artikelnummer
- 13581
- Online publication date
- 2021
- Status
- Published online
- Herausgeber
- Springer Science and Business Media LLC
- Herausgeber URL
- http://dx.doi.org/10.1038/s41598-021-92826-4
- Datum der Datenerfassung
- 2022
- Titel
- Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
- Ausgabe der Zeitschrift
- 11
Datenquelle: Crossref
- Abstract
- Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1-20.3), and intermediate precision ≤ 12.1% (95% CI 9.2-17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.
- Addresses
- Department of Sports Medicine, Rehabilitation and Disease Prevention, University of Mainz, Albert-Schweitzer Str. 22, 55128, Mainz, Germany.
- Autoren
- Elmo WI Neuberger
- Alexandra Brahmer
- Tobias Ehlert
- Katrin Kluge
- Keito FA Philippi
- Simone C Boedecker
- Julia Weinmann-Menke
- Perikles Simon
- DOI
- 10.1038/s41598-021-92826-4
- eISSN
- 2045-2322
- Externe Identifier
- PubMed Identifier: 34193884
- PubMed Central ID: PMC8245561
- Funding acknowledgements
- Johannes Gutenberg-Universität Mainz:
- Universtiy of Mainz Internal University Research Funding:
- Open access
- true
- ISSN
- 2045-2322
- Ausgabe der Veröffentlichung
- 1
- Zeitschrift
- Scientific reports
- Schlüsselwörter
- Humans
- Lupus Erythematosus, Systemic
- Adult
- Middle Aged
- Female
- Male
- Real-Time Polymerase Chain Reaction
- Cell-Free Nucleic Acids
- Sprache
- eng
- Medium
- Electronic
- Online publication date
- 2021
- Open access status
- Open Access
- Paginierung
- 13581
- Datum der Veröffentlichung
- 2021
- Status
- Published
- Publisher licence
- CC BY
- Datum der Datenerfassung
- 2021
- Titel
- Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients.
- Sub types
- Clinical Trial
- research-article
- Validation Study
- Journal Article
- Ausgabe der Zeitschrift
- 11
Files
https://www.nature.com/articles/s41598-021-92826-4.pdf https://europepmc.org/articles/PMC8245561?pdf=render
Datenquelle: Europe PubMed Central
- Abstract
- Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1-20.3), and intermediate precision ≤ 12.1% (95% CI 9.2-17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.
- Date of acceptance
- 2021
- Autoren
- Elmo WI Neuberger
- Alexandra Brahmer
- Tobias Ehlert
- Katrin Kluge
- Keito FA Philippi
- Simone C Boedecker
- Julia Weinmann-Menke
- Perikles Simon
- Autoren-URL
- https://www.ncbi.nlm.nih.gov/pubmed/34193884
- DOI
- 10.1038/s41598-021-92826-4
- eISSN
- 2045-2322
- Externe Identifier
- PubMed Central ID: PMC8245561
- Ausgabe der Veröffentlichung
- 1
- Zeitschrift
- Sci Rep
- Schlüsselwörter
- Adult
- Cell-Free Nucleic Acids
- Female
- Humans
- Lupus Erythematosus, Systemic
- Male
- Middle Aged
- Real-Time Polymerase Chain Reaction
- Sprache
- eng
- Country
- England
- Paginierung
- 13581
- PII
- 10.1038/s41598-021-92826-4
- Datum der Veröffentlichung
- 2021
- Status
- Published online
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2021
- Titel
- Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients.
- Sub types
- Clinical Trial
- Journal Article
- Validation Study
- Ausgabe der Zeitschrift
- 11
Datenquelle: PubMed
- Author's licence
- CC-BY
- Autoren
- Elmo WI Neuberger
- Alexandra Brahmer
- Tobias Ehlert
- Katrin Kluge
- Keito FA Philippi
- Simone C Boedecker
- Julia Weinmann-Menke
- Perikles Simon
- Hosting institution
- Universitätsbibliothek Mainz
- Sammlungen
- JGU-Publikationen
- Resource version
- Published version
- DOI
- 10.1038/s41598-021-92826-4
- File(s) embargoed
- false
- Open access
- true
- ISSN
- 2045-2322
- Zeitschrift
- Scientific reports
- Schlüsselwörter
- 610 Medizin
- 610 Medical sciences
- 796 Sport
- 796 Athletic and outdoor sports and games
- Sprache
- eng
- Open access status
- Open Access
- Paginierung
- 13581
- Datum der Veröffentlichung
- 2021
- Public URL
- https://openscience.ub.uni-mainz.de/handle/20.500.12030/7012
- Herausgeber
- Macmillan Publishers Limited, part of Springer Nature
- Datum der Datenerfassung
- 2022
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2022
- Zugang
- Public
- Titel
- Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
- Ausgabe der Zeitschrift
- 11
Files
validating_quantitative_pcr_a-20220519110437722.pdf
Datenquelle: OPENSCIENCE.UB
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