Comparison of paired cerebrospinal fluid and serum cell-free mitochondrial and nuclear DNA with copy number and fragment length.
Author(s): Chen A, Li J, Wang L, Huang Q, Zhu J, Wen S, Lyu J, Wu W
Publication: J Clin Lab Anal, 2020, Vol. 34, Page e23238
PubMed ID: 32052892 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare the fragment size of mitochondrial and nuclear cell-free DNA (cfDNA) between cerebrospinal fluid (CSF) and serum directly and after cfDNA extraction. Protein, potassium, and sodium levels were also compared between serum and CSF.
Conclusion of Paper
Serum had much higher concentrations of cfDNA than CSF but levels of sodium and potassium were comparable in serum and CSF. Bioanalyzer electropherograms showed a high baseline in raw CSF and serum but distinct peaks were still identifiable and dilution decreased background. Extracted cfDNA from CSF and serum had distinct fragment size profiles. While all products were amplifiable directly from CSF and serum, the standard deviations in copy numbers were smaller when extraction was performed prior to amplification, especially for serum. As expected, there were more copies of the shorter amplicons than longer amplicons, indicating that some of the cfDNA was small. Serum had more copies of the small and medium size fragments of mitochondrial (ND1) cfDNA than CSF but fewer copies of the longest fragment. In contrast, there were comparable copy numbers of the small fragment of nuclear (GAPDH) DNA in serum and CSF but more copies of the medium and long fragments of nuclear DNA in CSF than serum. Both serum and plasma had comparable numbers of copies of the short amplicons of nuclear and mitochondrial cfDNA, but the longest amplicons of mitochondrial DNA were more abundant than nuclear DNA in CSF and serum, indicating that mitochondrial DNA is more intact. Quantified copies of cfDNA were strongly correlated between real-time PCR and ddPCR.
Studies
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Study Purpose
The purpose of this study was to compare the fragment size of mitochondrial and nuclear cfDNA between CSF and serum directly and after cfDNA extraction. Protein, potassium, and sodium levels were also compared between serum and CSF. Blood and CSF were collected simultaneously from seven patients with cerebral hemorrhage, brain trauma, intracranial hypertension, or fever and centrifuged at 1600 x g at 4°C for 10 min. The serum and CSF supernatant were transferred to new tubes and centrifuged at 16000 x g for 10 min at 4°C to remove the remaining cellular debris. CSF and serum were aliquoted and stored at -80°C. Proteins were quantified using the BCA protein assay. Sodium and potassium were quantified by PFP7 flame spectrophotometer. DNA was extracted using the TIANamp Micro DNA Kit. cfDNA fragment size was evaluated by bioanalyzer and by real-time PCR amplification of 61, 168, and 241 bp fragments of GAPDH (nuclear cfDNA) and 57, 167, and 240 bp fragments of ND1 (mitochondrial cfDNA) in extracted cFDNA as well as in serum and CSF. cfDNA copy number was evaluated by ddPCR amplification of the 61 bp fragment of GAPDH.
Summary of Findings:
Serum had much higher concentrations of cfDNA than CSF (P<0.001), but levels of sodium and potassium in serum and CSF were comparable. Bioanalyzer analysis showed a high baseline in raw CSF and serum but distinguishable peaks were still observed in CSF at 60 bp and 1495-7022 bp and in serum at 1680 bp and 10380 bp. Dilution by 5-fold decreased the background noise. Extracted cfDNA from CSF had peaks at 178 bp and from 338-8185 bp while cfDNA extracted from serum had peaks at 199 bp and a series of peaks 360-4949 bp. While all products were amplifiable directly from CSF and serum, the standard deviations in copy numbers were smaller when extraction was performed prior to amplification especially for serum. As expected, there were more copies of the shorter amplicons than longer amplicons, indicating that some of the cfDNA was small. Serum had more copies of the 57 bp and 167 bp fragments of mitochondrial cfDNA than CSF (P=0.0030 and P=0.0455, respectively) but fewer copies of the longest fragment (P=0.0479). In contrast, there were comparable copies of the small fragment of nuclear DNA in serum and CSF but more copies of the medium and long fragments of nuclear DNA in CSF than serum (P=0.0336 and P=0.0122, respectively). Both serum and plasma had comparable numbers of copies of the short amplicons of nuclear and mitochondrial cfDNA, but serum had more copies of the mid-size fragments of mitochondrial DNA than nuclear DNA (P=0.0025). There were more copies of the 240 bp fragment of mitochondrial DNA than the 241 bp fragment of nuclear DNA in CSF (P=0.0104) and serum (P=0.0010). Quantified copies of cfDNA were strongly correlated between real-time PCR and ddPCR (R2=0.872, P<0.0001).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Real-time qPCR Electrolyte/Metal Flame atomic absorption spectrometry Protein Colorimetric assay DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location CSF
Serum
Analyte Extraction and Purification Analyte isolation method No isolation performed
DNA isolated with TIANamp Micro DNA Kit
Real-time qPCR Specific Targeted nucleic acid ND1 (mitochondrial DNA)
GAPDH (nuclear DNA)
Real-time qPCR Specific Length of gene fragment 57 bp
61 bp
167 bp
168 bp
240 bp
241 bp
Automated electrophoresis/Bioanalyzer Specific Template modification Undiluted
Diluted 5-fold