A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers

Takousis, Petros; Devonshire, Alison S.; Redshaw, Nicholas; Baumgarten, Louisa von; Whale, Alexandra S.; Jones, Gerwyn M.; Fernandez-Gonzalez, Ana; Martin, Jan; Foy, Carole A.; Alexopoulos, Panagiotis; Huggett, Jim F. und Perneczky, Robert (2022): A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers. In: New Biotechnology, Bd. 72: S. 97-106

Abstract

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer's disease (AD). However, the in-fluence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap (R) DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin ; Medizin > Munich Cluster for Systems Neurology (SyNergy)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 1871-6784
• Sprache: Englisch
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 390857198