Comparison of paired cerebrospinal fluid and serum cell-free mitochondrial and nuclear DNA with copy number and fragment length

Abstract

Background: Most studies on cell-free DNA (cfDNA) were only for single body fluids; however, the differences in cfDNA distribution between two body fluids are rarely reported. Hence, in this work, we compared the differences in cfDNA distribution between cerebrospinal fluid (CSF) and serum of patients with brain-related diseases.

Methods: The fragment length of cfDNA was determined by using Agilent 2100 Bioanalyzer. The copy numbers of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) were determined by using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) with three pairs of mitochondrial ND1 and nuclear GAPDH primers, respectively.

Results: There were short (~60 bp), medium (~167 bp), and long (>250 bp) cfDNA fragment length distributions totally obtained from CSF and serum using Agilent 2100 Bioanalyzer. The results of both qPCR and ddPCR confirmed the existence of these three cfDNA fragment ranges in CSF and serum. According to qPCR, the copy numbers of long cf-mtDNA, medium, and long cf-nDNA in CSF were significantly higher than in paired serum. In CSF, only long cf-mtDNA's copy numbers were higher than long cf-nDNA. But in serum, the copy numbers of medium and long cf-mtDNA were higher than the corresponding cf-nDNA.

Conclusion: The cf-nDNA and cf-mtDNA with different fragment lengths differentially distributed in the CSF and serum of patients with brain disorders, which might serve as a biomarker of human brain diseases.

Keywords: cell-free mitochondrial DNA; cell-free nuclear DNA; cerebrospinal fluid; copy number; fragment length; serum.

Figure 1

Evaluation of cfDNA fragment length using Agilent 2100 Bioanalyzer. A‐D, Characterization results of raw CSF with fivefold dilution (A), raw serum with fivefold dilution (B), extracted CSF (C) and extracted serum (D) from patient 3. X‐axis represents the migration time of DNA fragments. Y‐axis indicates fluorescence intensity. The lower marker is 35 bp, and the upper marker is 10 380 bp. The numbers above peaks indicate the length of DNA fragments

Figure 2

Quantification of cfDNA copy number by qPCR. A, B, Raw CSF was amplified with primer pairs of ND1 (A) and GAPDH (B). C, D, Extracted CSF was amplified with primer pairs of ND1 (C) and GAPDH (D). E, F, Extracted serum was amplified with primer pairs of ND1 (E) and GAPDH (F). X‐axis represents patients, and Y‐axis represents log₁₀ of copies/µL in initial raw samples. Data are presented as mean ± SD, n = 3

Figure 3

Comparison of copy number of cfDNA in short fragment length by qPCR. A, B, The copy numbers of S‐cf‐mtDNA (A) and S‐cf‐nDNA (B) in CSF and serum. C, D, Copy numbers of S‐cf‐mtDNA and S‐cf‐nDNA in CSF (C) and serum (D). **P ˂ .01. Data are presented as mean ± SD, n = 7

Figure 4

Comparison of copy number of cfDNA in medium fragment length by qPCR. A, B, The copy numbers of M‐cf‐mtDNA (A) and M‐cf‐nDNA (B) in CSF and serum. C, D, Copy numbers of M‐cf‐mtDNA and M‐cf‐nDNA in CSF (C) and serum (D). *P ˂ .05, **P ˂ .01. Data are presented as mean ± SD, n = 7

Figure 5

Comparison of copy number of cfDNA in long fragment length by qPCR. A, B, The copy numbers of L‐cf‐mtDNA (A) and L‐cf‐nDNA (B) in CSF and serum. C, D, Copy numbers of L‐cf‐mtDNA and L‐cf‐nDNA in CSF (C) and serum (D). *P ˂ .05, **P ˂ .01. Data are presented as mean ± SD, n = 7

Figure 6

Quantification of cfDNA copy number by ddPCR and comparison with qPCR. A, B, The overall trend (A) and liner correlation (B) between qPCR and ddPCR. All detection values of each method represent that all seven patients' extracted CSF and serum were amplified by all six primer pairs (n = 84), ****P ˂ .0001. C, D, Extracted CSF was amplified with primer pairs of ND1 (C) and GAPDH (D). E, F, Extracted serum was amplified with primer pairs of ND1 (E) and GAPDH (F). X‐axis represents primer pairs, and Y‐axis represents log₁₀ of copies/µL in initial raw samples. *P ˂ .05, **P ˂ .01, ***P ˂ .001. Data are presented as mean ± SD, n = 7