Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid.
Author(s): Song HH, Park H, Cho D, Bang HI, Oh HJ, Kim J
Publication: Ann Lab Med, 2024, Vol. 44, Page 294-298
PubMed ID: 38151854 PubMed Review Paper? No
Purpose of Paper
This paper sought to optimize the extraction of cell-free (cfDNA) DNA from cerebrospinal fluid (CSF) by comparing yields obtained using the Cobas cfDNA Sample Preparation Kit with either a single 100 µL or two 50 µL elutions with those obtained using the Chemagic cfDNA 2k Kit H24 with 50 μL, 75 μL, or 100 μL of beads. The effect of vacuum concentration and the use of poly(A) RNA buffer were also examined. The count and variant allele frequency of spiked-in mutations were also compared between the optimized Cobas and Chemagic methods.
Conclusion of Paper
Using artificial CSF specimens, cfDNA yield was significantly higher when two-50 µL elution steps were used with the column-based Cobas kit rather than when a single 100 µL elution was used and when a vacuum step was included. cfDNA yield from the magnetic bead-based Chemagic kit was highest when 75 μL of beads were used rather than 50 or 100 μL and when a vacuum step was included (P<0.0001). Importantly, the concordance rate for the average size of cfDNA between vacuum-concentrated and non- concentrated specimens was 0.78. When the optimized protocol for each kit was used, the average percent recovery was significantly higher using the Chemagic kit with 75 μL beads than the Cobas kit with a two-step elution. The average number of IDH1 R132C droplets was higher using the Chemagic kit than the Cobas kit, but the variant allele frequency was similar between the two extraction methods. Consequently, the Chemagic kit was chosen for subsequent experiments using clinical specimens. In clinical CSF samples, the yield of cfDNA isolated with the Chemagic kit and 75 μL of beads was not affected by the addition of poly(A) RNA buffer.
Studies
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Study Purpose
This study sought to optimize the extraction of cfDNA from CSF by comparing yields obtained using the Cobas cfDNA Sample Preparation kit with either a single 100 µL or two 50 µL elutions with those obtained using the Chemagic cfDNA 2k Kit H24 with 50 μL, 75 μL, or 100 μL of beads. The effect of vacuum concentration and the use of poly(A) RNA buffer were also examined. The count and variant allele frequency of spiked-in mutations were also compared between the optimized Cobas and Chemagic methods. A total of 59 artificial CSF specimens were generated by adding a ctDNA master mix at an allele frequency of 2% to remnant, non-bloody lumbar puncture CSF specimens from the archive. DNA was isolated from 29 of these artificial CSF specimens using the column-based Cobas cfDNA Sample Preparation Kit with a single 100 µL or two 50 µL elutions and from 30 of these artificial specimens using the magnetic bead-based Chemagic cfDNA 2k Kit H24 method using 50 μL, 75 μL, or 100 μL of beads. cfDNA was concentrated using a HyperVACMAX VC2200 centrifugal vacuum concentrator. Additionally, five CSF specimens were collected by lumbar puncture from patients (diagnosis not specified), and RNA was extracted using the Chemagic cfDNA 2k Kit H24 method using 75 μL of beads with and without the addition of poly(A) RNA buffer. DNA was quantified by Screentape and individual mutations were detected by droplet digital PCR.
Summary of Findings:
Using artificial CSF specimens, the yield of cfDNA that was isolated using the column-based Cobas kit was significantly higher when two-50 µL elution steps were used rather than a single 100 µL elution and when a vacuum step was used (P<0.0001). The yield of cfDNA isolated using the magnetic bead-based Chemagic kit was highest when 75 μL of beads were used rather than 50 or 100 μL (P<0.001) and when a vacuum step was included (P<0.0001). Importantly, the concordance rate for the average size of cfDNA between vacuum-concentrated and non-concentrated specimens was 0.78. Using the optimized protocol for each kit, the average percent recovery was significantly higher using the Chemagic kit with 75 μL beads than the Cobas kit with a two-step elution (162.3±47.7 versus 82.5±19.2, P<0.001), and the coefficient of variance (CV) for each method was comparable (23.3% versus 29.4%). The average number of IDH1 R132C droplets was higher when the Chemagic kit was used rather than the Cobas kit (1,855 versus 517.4, P<0.0001), but the variant allele frequency was similar between the two extraction methods (0.4% versus 0.5%). Consequently, the Chemagic kit was chosen for the subsequent of clinical specimens. In clinical CSF samples, the yield of cfDNA that was isolated using the Chemagic kit with 75 μL of beads was not affected by the addition of poly(A) RNA buffer.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte purification Chemagic cfDNA 2k Kit H24 method using 50 μL beads
Chemagic cfDNA 2k Kit H24 method using 75 μL beads
Chemagic cfDNA 2k Kit H24 method using 100 μL beads
Cobas cfDNA Sample Preparation kit with a single 100 µL elution
Cobas cfDNA Sample Preparation kit with two 50 µL elation steps
Chemagic cfDNA 2k Kit H24 method using 75 μL beads and poly(A) RNA buffer
Chemagic cfDNA 2k Kit H24 method using 75 μL beads without poly(A) RNA buffer
Automated electrophoresis/Bioanalyzer Specific Template modification Vacuum concentrated
Not vacuum concentrated