Please use this identifier to cite or link to this item:
http://doi.org/10.25358/openscience-7000
Authors: | Neuberger, Elmo W. I. Brahmer, Alexandra Ehlert, Tobias Kluge, Katrin Philippi, Keito F. A. Boedecker, Simone C. Weinmann-Menke, Julia Simon, Perikles |
Title: | Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients |
Online publication date: | 19-May-2022 |
Year of first publication: | 2021 |
Language: | english |
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. |
DDC: | 610 Medizin 610 Medical sciences 796 Sport 796 Athletic and outdoor sports and games |
Institution: | Johannes Gutenberg-Universität Mainz |
Department: | FB 02 Sozialwiss., Medien u. Sport FB 04 Medizin |
Place: | Mainz |
ROR: | https://ror.org/023b0x485 |
DOI: | http://doi.org/10.25358/openscience-7000 |
Version: | Published version |
Publication type: | Zeitschriftenaufsatz |
License: | CC BY |
Information on rights of use: | https://creativecommons.org/licenses/by/4.0/ |
Journal: | Scientific reports 11 |
Pages or article number: | 13581 |
Publisher: | Macmillan Publishers Limited, part of Springer Nature |
Publisher place: | London |
Issue date: | 2021 |
ISSN: | 2045-2322 |
Publisher DOI: | 10.1038/s41598-021-92826-4 |
Appears in collections: | JGU-Publikationen |
Files in This Item:
File | Description | Size | Format | ||
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validating_quantitative_pcr_a-20220519110437722.pdf | 2.31 MB | Adobe PDF | View/Open |