Analysis of urine cell-free DNA copy number and fragment size from healthy individuals.

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.