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Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma

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 kno...

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Autores principales: Breitbach, Sarah, Tug, Suzan, Helmig, Susanne, Zahn, Daniela, Kubiak, Thomas, Michal, Matthias, Gori, Tommaso, Ehlert, Tobias, Beiter, Thomas, Simon, Perikles
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940427/
https://www.ncbi.nlm.nih.gov/pubmed/24595313
http://dx.doi.org/10.1371/journal.pone.0087838
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author Breitbach, Sarah
Tug, Suzan
Helmig, Susanne
Zahn, Daniela
Kubiak, Thomas
Michal, Matthias
Gori, Tommaso
Ehlert, Tobias
Beiter, Thomas
Simon, Perikles
author_facet Breitbach, Sarah
Tug, Suzan
Helmig, Susanne
Zahn, Daniela
Kubiak, Thomas
Michal, Matthias
Gori, Tommaso
Ehlert, Tobias
Beiter, Thomas
Simon, Perikles
author_sort Breitbach, Sarah
collection PubMed
description 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.
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spelling pubmed-39404272014-03-06 Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma Breitbach, Sarah Tug, Suzan Helmig, Susanne Zahn, Daniela Kubiak, Thomas Michal, Matthias Gori, Tommaso Ehlert, Tobias Beiter, Thomas Simon, Perikles PLoS One Research Article 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. Public Library of Science 2014-03-03 /pmc/articles/PMC3940427/ /pubmed/24595313 http://dx.doi.org/10.1371/journal.pone.0087838 Text en © 2014 Breitbach et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Breitbach, Sarah
Tug, Suzan
Helmig, Susanne
Zahn, Daniela
Kubiak, Thomas
Michal, Matthias
Gori, Tommaso
Ehlert, Tobias
Beiter, Thomas
Simon, Perikles
Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma
title Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma
title_full Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma
title_fullStr Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma
title_full_unstemmed Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma
title_short Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma
title_sort direct quantification of cell-free, circulating dna from unpurified plasma
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940427/
https://www.ncbi.nlm.nih.gov/pubmed/24595313
http://dx.doi.org/10.1371/journal.pone.0087838
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