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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
Version: Published version
Publication type: Zeitschriftenaufsatz
License: CC BY
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Journal: Scientific reports
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

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