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.
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| Title: | 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. |
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| Authors: | Takousis, Petros1 (AUTHOR), Devonshire, Alison S.1,2 (AUTHOR) alison.devonshire@lgcgroup.com, Redshaw, Nicholas2 (AUTHOR), von Baumgarten, Louisa3,4 (AUTHOR), Whale, Alexandra S.2 (AUTHOR), Jones, Gerwyn M.2 (AUTHOR), Fernandez-Gonzalez, Ana2 (AUTHOR), Martin, Jan5 (AUTHOR), Foy, Carole A.2 (AUTHOR), Alexopoulos, Panagiotis6,7 (AUTHOR), Huggett, Jim F.2,8 (AUTHOR), Perneczky, Robert1,9,10,11,12 (AUTHOR) |
| Source: | New Biotechnology. Dec2022, Vol. 72, p97-106. 10p. |
| Subjects: | Cell-free DNA, Alzheimer's disease, Brain diseases, Mitochondrial DNA, Brain cancer, Plant mitochondria, DNA, Cerebrospinal fluid, Brain tumors |
| 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 influence 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® 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. [Display omitted] • Approaches to cell-free DNA biomarkers in cerebrospinal fluid vary between studies. • Kits for cell-free DNA (cfDNA) extraction from cerebrospinal fluid were compared. • Spike-in recovery correlated with mitochondrial and nuclear genomic cfDNA yield. • Sensitive assays to repetitive elements are required for nuclear cfDNA detection. • Ratio of mitochondrial to nuclear cfDNA is a potential normalisation strategy. [ABSTRACT FROM AUTHOR] |
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| Database: | Engineering Source |
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