Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR.
To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ0) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA. In the results, the ddPCR method showed high sensitivity and specificity. When the DNA was extracted from plasma prior to ddPCR, the ccf-mtDNA copy number was higher than that measured without extraction. This difference was not due to a PCR inhibitor, such as EDTA-Na2, an anti-coagulant in plasma, because standard EDTA-Na2concentration (5 mM) did not significantly inhibit ddPCR reactions. The difference might be attributable to plasma exosomal mtDNA, which was 4.21 ± 0.38 copies/μL of plasma, accounting for ∼19% of plasma ccf-mtDNA. Therefore, ddPCR can quickly and reliably detect ccf-mtDNA from plasma with a prior DNA extraction step, providing for a more accurate detection of ccf-mtDNA. The direct use of plasma as a template in ddPCR is suitable for the detection of exogenous cell-free nucleic acids within plasma, but not of nucleic acids that have a vesicle-associated form, such as exosomal mtDNA. Graphical Abstract Designs of the present work. *: Module 1, #: Module 2, &: Module 3.
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
Wei
W
Ye
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
Xiaojun
X
Tang
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
Chu
C
Liu
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
Chaowei
C
Wen
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
Wei
W
Li
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China. ljxwzmc@aliyun.com.
Jianxin
J
Lyu
D000328:Adult; D000073888:Cell-Free Nucleic Acids / Q000032:analysis* / Q000097:blood / Q000235:genetics; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000097:blood / Q000235:genetics; D005240:Feasibility Studies; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D008928:Mitochondria / Q000235:genetics*; D010949:Plasma / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D055815:Young Adult
D000073888:Cell-Free Nucleic Acids; D004272:DNA, Mitochondrial
Circulating cell-free DNA; Droplet digital PCR; Mitochondrial DNA