Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid
- Abstract
- A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic bead-based (MB) isolation. Furthermore, we modified both methods, including pre- and post-elution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P<0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.
- All Author(s)
- Ho Hyun Song
; Hyeran Park
; Doohwan Cho
; Hae In Bang
; Hyuk-Jin Oh
; Jieun Kim
- Issued Date
- 2024
- Type
- Article
- Keyword
- Cell-free nucleic acids; Cerebrospinal fluid; Circulating tumor DNA; Droplet digital PCR; Vacuum
- Publisher
- 대한진단검사의학회
- ISSN
- 2234-3806
; 2234-3814
- Citation Title
- Annals of laboratory medicine
- Citation Volume
- 44
- Citation Number
- 3
- Citation Start Page
- 294
- Citation End Page
- 298
- Language(ISO)
- eng
- DOI
- 10.3343/alm.2023.0267
- URI
- http://schca-ir.schmc.ac.kr/handle/2022.oak/1314
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