title: Direct quantification of cell-free, circulating DNA from unpurified plasma.
abstract: Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.
authors.affiliation: Department of Sports Medicine, Rehabilitation and Prevention, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Breitbach
authors.affiliation: Department of Sports Medicine, Rehabilitation and Prevention, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Suzan
authors.initials: S
authors.lastname: Tug
authors.affiliation: Department of Sports Medicine, Rehabilitation and Prevention, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Helmig
authors.affiliation: Department of Health Psychology, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Zahn
authors.affiliation: Department of Health Psychology, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kubiak
authors.affiliation: Department of Psychosomatic Medicine and Psychotherapy, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Michal
authors.affiliation: Department of Cardiology, Angiology and Internal Medicine, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Gori
authors.affiliation: Department of Sports Medicine, Rehabilitation and Prevention, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Ehlert
authors.affiliation: Department of Sports Medicine, Medical Clinic, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Beiter
authors.affiliation: Department of Sports Medicine, Rehabilitation and Prevention, Johannes Gutenberg-University of Mainz, Mainz, Germany.
authors.forename: Perikles
authors.initials: P
authors.lastname: Simon
mesh_terms: D001483:Base Sequence; D016022:Case-Control Studies; D002474:Cell-Free System; D003327:Coronary Disease / Q000097:blood / Q000235:genetics; D004247:DNA / Q000097:blood*; D017931:DNA Primers; D015444:Exercise; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004247:DNA