An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.
J Biol Chem
; 295(46): 15677-15691, 2020 11 13.
Article
em En
| MEDLINE
| ID: mdl-32900851
ABSTRACT
Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of â¼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Mitocondrial
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Núcleo Celular
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Ensaios de Triagem em Larga Escala
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Ácidos Nucleicos Livres
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Mitocôndrias
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos