Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia
Background Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. Methods and Results Newborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40–45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for β-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. Conclusions Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses..
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Artikel |
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Erscheinungsjahr: |
2022 |
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Erschienen: |
2022 |
Enthalten in: |
Zur Gesamtaufnahme - volume:50 |
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Enthalten in: |
Molecular biology reports - 50(2022), 2 vom: 13. Dez., Seite 1533-1544 |
Sprache: |
Englisch |
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Beteiligte Personen: |
Bitenc, Marie [VerfasserIn] |
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Links: |
Volltext [lizenzpflichtig] |
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BKL: | |
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Themen: |
Cell-free DNA |
Anmerkungen: |
© The Author(s) 2022 |
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doi: |
10.1007/s11033-022-08135-0 |
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funding: |
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Förderinstitution / Projekttitel: |
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PPN (Katalog-ID): |
OLC2133682546 |
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520 | |a Background Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. Methods and Results Newborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40–45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for β-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. Conclusions Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses. | ||
650 | 4 | |a Cell-free DNA | |
650 | 4 | |a Digital PCR | |
650 | 4 | |a Mitochondrial cfDNA | |
650 | 4 | |a Nuclear cfDNA | |
650 | 4 | |a Perinatal asphyxia | |
650 | 4 | |a qRT-PCR | |
700 | 1 | |a Grebstad Tune, Benedicte |4 aut | |
700 | 1 | |a Melheim, Maria |4 aut | |
700 | 1 | |a Atneosen-Åsegg, Monica |4 aut | |
700 | 1 | |a Lai, Xiaoran |4 aut | |
700 | 1 | |a Rajar, Polona |4 aut | |
700 | 1 | |a Solberg, Rønnaug |4 aut | |
700 | 1 | |a Baumbusch, Lars Oliver |0 (orcid)0000-0003-1290-312X |4 aut | |
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