Autoren:	Neuberger, Elmo W. I. Brahmer, Alexandra Ehlert, Tobias Kluge, Katrin Philippi, Keito F. A. Boedecker, Simone C. Weinmann-Menke, Julia Simon, Perikles
Titel:	Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
Online-Publikationsdatum:	19-Mai-2022
Erscheinungsdatum:	2021
Sprache des Dokuments:	Englisch
Zusammenfassung/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-Sachgruppe:	610 Medizin 610 Medical sciences 796 Sport 796 Athletic and outdoor sports and games
Veröffentlichende Institution:	Johannes Gutenberg-Universität Mainz
Organisationseinheit:	FB 02 Sozialwiss., Medien u. Sport FB 04 Medizin
Veröffentlichungsort:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-7000
Version:	Published version
Publikationstyp:	Zeitschriftenaufsatz
Nutzungsrechte:	CC BY
Informationen zu den Nutzungsrechten:	https://creativecommons.org/licenses/by/4.0/
Zeitschrift:	Scientific reports 11
Seitenzahl oder Artikelnummer:	13581
Verlag:	Macmillan Publishers Limited, part of Springer Nature
Verlagsort:	London
Erscheinungsdatum:	2021
ISSN:	2045-2322
DOI der Originalveröffentlichung:	10.1038/s41598-021-92826-4
Enthalten in den Sammlungen:	JGU-Publikationen