Clinica chimica acta; international journal of clinical chemistry · 2025
Analysis of urine cell-free DNA copy number and fragment size from healthy individuals.
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Wang Jufen, Wu Zilong, Wu Bingyu, Lin Xinghao, Wu Wenhe, Li Jun
Аннотация
OBJECTIVE: This study characterizes urine cell-free DNA (cfDNA) copy number and fragment size in healthy individuals and explores their associations with routine clinical parameters.
METHODS: Sixty healthy subjects were enrolled, providing paired blood and urine samples. Six primer pairs targeting nuclear (GAPDH-61/168/241) and mitochondrial DNA (ND1-57/167/240) were designed for absolute qPCR. Optimal urine collection, pre-treatment, and cfDNA detection protocols were evaluated. Correlations between cfDNA characteristics (fragment size and copy number) and clinical parameters (complete blood count, urinalysis, hepatic/renal function biomarkers) were analyzed.
RESULTS: Non-extracted urine retained a higher proportion of fragments < 100 bp and > 2000 bp than extracted samples. The optimal pre-treatment involved immediate EDTA addition, centrifugation at 4°C, and storage at -80°C. Urine cfDNA comprised short, medium, and long fragments. Cell-free mitochondrial DNA (cf-mtDNA) showed the highest copy numbers in short fragments, decreasing with length, whereas cell-free nuclear DNA (cf-nDNA) peaked in medium fragments. ND1-57 Cq values correlated negatively with neutrophil percentage (P < 0.01) and positively with lymphocyte percentage (P < 0.05). Lymphocyte percentage was moderately correlated with ND1 short fragment (ND1-S, P < 0.01) and weakly with the ND1-S to ND1 medium fragment (ND1-M) ratio (P < 0.05). Absolute lymphocyte count correlated weakly with ND1-S (P < 0.01) and ND1-M (P < 0.05). Neutrophil percentage correlated weakly with ND1-S (P < 0.01) and ND1-S to ND1 long fragment (ND1-L) ratio (P < 0.05).
CONCLUSION: Urine cfDNA displays three distinct fragment sizes, with cf-mtDNA predominantly found in short fragments and showing stronger associations with physiological parameters than cf-nDNA.
METHODS: Sixty healthy subjects were enrolled, providing paired blood and urine samples. Six primer pairs targeting nuclear (GAPDH-61/168/241) and mitochondrial DNA (ND1-57/167/240) were designed for absolute qPCR. Optimal urine collection, pre-treatment, and cfDNA detection protocols were evaluated. Correlations between cfDNA characteristics (fragment size and copy number) and clinical parameters (complete blood count, urinalysis, hepatic/renal function biomarkers) were analyzed.
RESULTS: Non-extracted urine retained a higher proportion of fragments < 100 bp and > 2000 bp than extracted samples. The optimal pre-treatment involved immediate EDTA addition, centrifugation at 4°C, and storage at -80°C. Urine cfDNA comprised short, medium, and long fragments. Cell-free mitochondrial DNA (cf-mtDNA) showed the highest copy numbers in short fragments, decreasing with length, whereas cell-free nuclear DNA (cf-nDNA) peaked in medium fragments. ND1-57 Cq values correlated negatively with neutrophil percentage (P < 0.01) and positively with lymphocyte percentage (P < 0.05). Lymphocyte percentage was moderately correlated with ND1 short fragment (ND1-S, P < 0.01) and weakly with the ND1-S to ND1 medium fragment (ND1-M) ratio (P < 0.05). Absolute lymphocyte count correlated weakly with ND1-S (P < 0.01) and ND1-M (P < 0.05). Neutrophil percentage correlated weakly with ND1-S (P < 0.01) and ND1-S to ND1 long fragment (ND1-L) ratio (P < 0.05).
CONCLUSION: Urine cfDNA displays three distinct fragment sizes, with cf-mtDNA predominantly found in short fragments and showing stronger associations with physiological parameters than cf-nDNA.