Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia
© 2022. The Author(s)..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:50 |
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| Enthalten in: |
Molecular biology reports - 50(2023), 2 vom: 13. Feb., Seite 1533-1544 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Bitenc, Marie [VerfasserIn] |
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| Links: |
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| Themen: |
Cell-Free Nucleic Acids |
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| Anmerkungen: |
Date Completed 02.02.2023 Date Revised 02.09.2023 published: Print-Electronic Citation Status MEDLINE |
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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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| 100 | 1 | |a Bitenc, Marie |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia |
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| 500 | |a published: Print-Electronic | ||
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| 520 | |a © 2022. The Author(s). | ||
| 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 Journal Article | |
| 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 | |
| 650 | 7 | |a Cell-Free Nucleic Acids |2 NLM | |
| 700 | 1 | |a Grebstad Tune, Benedicte |e verfasserin |4 aut | |
| 700 | 1 | |a Melheim, Maria |e verfasserin |4 aut | |
| 700 | 1 | |a Atneosen-Åsegg, Monica |e verfasserin |4 aut | |
| 700 | 1 | |a Lai, Xiaoran |e verfasserin |4 aut | |
| 700 | 1 | |a Rajar, Polona |e verfasserin |4 aut | |
| 700 | 1 | |a Solberg, Rønnaug |e verfasserin |4 aut | |
| 700 | 1 | |a Baumbusch, Lars Oliver |e verfasserin |4 aut | |
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