Analysis of urine cell-free DNA copy number and fragment size from healthy individuals.
Author(s): Wang J, Wu Z, Wu B, Lin X, Wu W, Li J
Publication: Clin Chim Acta, 2025, Vol. 579, Page 120627
PubMed ID: 40998077 PubMed Review Paper? No
Purpose of Paper
This paper compared cell-free (cfDNA) size profiles in case-matched plasma and urine from healthy volunteers following DNA extraction or direct amplification without extraction. Additionally, the size profiles and copy numbers of nuclear (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH) and mitochondrial (ND1) cfDNA were compared among urine specimens that were frozen immediately with and without EDTA and stored with and without EDTA at room temperature and 4°C for 0 h, 1.5 h, 3 h and 5 h before freezing. Potential correlations between the copy number of amplicons of different sizes and clinical parameters, including blood cell counts, urinalysis results, and levels of hepatic/renal function biomarkers, were investigated.
Conclusion of Paper
When cfDNA was directly amplified (i.e., no DNA extraction step was performed), minor peaks at 178 bp and 62 bp were observed in plasma and urine electropherograms, respectively, but these peaks were not observed when DNA extraction was performed. Without ligation and amplification, cfDNA was not observable by gel electrophoresis regardless of whether DNA extraction was performed. When ligation and amplification were performed, a cfDNA smear was visible from extracted specimens but lacked shorter (<100 bp) and longer (>2,000 bp) fragments, which were visible among specimens that did not undergo extraction.
cfDNA concentration was comparable in urine when frozen at -80°C immediately or after the addition of EDTA; when stored at 4°C for 1.5, 3 or 5 h prior to the addition of EDTA; and when stored at 4°C for 3 h with and without EDTA. However, cfDNA yield was affected when urine was stored at room temperature for 1.5, 3 or 5 h before EDTA was added compared to 0 h (P<0.001), when stored at room temperature for 3 h compared to specimens without EDTA (P<0.001), and when stored without EDTA for 3 h at room temperature compared to matched controls stored at 4°C (P=0.003). The authors state that cfDNA fragment size was significantly affected by storage at 4°C or room temperature and by the addition of EDTA; consequently, they recommend adding EDTA immediately after urine collection, two rounds of centrifugation at 4°C, and then immediately freezing the supernatant at -80°C (data not shown). The mean copy numbers of long, medium and short amplicons of mitochondrial ND1 (94.84, 288.11, and 1084.98 copies/μL, respectively) were much higher than those of nuclear GAPDH (4.46, 15.53, and 6.32 copies/μL, respectively) in urine. The authors highlight that the copy numbers of ND1 decrease with increasing fragment size while GAPDH copies remain more consistent, with the highest numbers of ND1 observed for the medium length amplicon. The authors state there were no associations between GAPDH copy number and clinical parameters (blood cell counts, urinalysis results, and levels of hepatic/renal function biomarkers) in an initial analysis. However, the quantification cycle values (Cq) of the short amplicon of ND1 were correlated with lymphocyte percentage (Pearson r = -0.391, P = 0.002), neutrophil percentage (Pearson r =-0.344, P = 0.007), and the absolute value of lymphocyte counts (Spearman ρ = 0.364, P = 0.004). The copy number of the ND1 57 bp amplicon was correlated with pH (Spearman ρ = 0.258, P = 0.047), neutrophil percentage (Spearman ρ = 0.352, P = 0.006), urine specific gravity (Spearman ρ = -0.256, P = 0.048), absolute value lymphocyte counts (Spearman ρ = -0.397, P = 0.002), lymphocyte percentage (Spearman ρ = -0.410, P = 0.001). Additionally the copies of ND1 that were 57 -167 bp (ND1-S) were correlated with neutrophil percentage (Spearman ρ = 0.397, P = 0.002), lymphocyte percentage (Spearman ρ = -0.345, P = 0.001) and absolute lymphocyte count (Spearman ρ = -0.388, P = 0.002), and copies of ND1 between 167 and 240 bp (ND1-M) were correlated with absolute lymphocyte count (Spearman ρ = -0.306, P = 0.017). The ND1-S to ND1-M ratio was correlated with percentage neutrophils (Spearman ρ = -0.368, P = 0.004) and lymphocytes (Spearman ρ = 0.321, P = 0.012).
Studies
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Study Purpose
This study compared cell-free (cfDNA) size profiles in case-matched plasma and urine from healthy volunteers following DNA extraction or direct amplification without extraction. Additionally, size profiles and copy numbers of nuclear (GAPDH) and mitochondrial (ND1) cfDNA were compared among urine specimens that were frozen immediately with and without EDTA and stored with and without EDTA at room temperature and 4°C for 0 h, 1.5 h, 3 h and 5 h before freezing. Potential correlations between the copy number of differently-sized amplicons and clinical parameters, including blood cell counts, urinalysis results, and levels of hepatic/renal function biomarkers, were also investigated. Blood (anticoagulant not specified) and urine were collected from sixty healthy individuals (aged 21–40 years; 57% male and 43% female). Plasma was obtained by centrifugation at 1600 ×g for 10 min at 4°C followed by 16,000 ×g for 10 min at 4°C and stored frozen. Unless otherwise specified, EDTA was added to urine, urine was centrifuged (details not provided), and supernatants were stored at -80°C. cfDNA was extracted from plasma and urine with the MiniMax High-Efficiency cfDNA Isolation Kit. The fragment size profile was analyzed with the Agilent 4150 TapeStation system and by gel electrophoresis. cfDNA was quantified by amplification of multiple differently-sized fragments of mitochondrial ND1 (57, 167 and 240 bp) and nuclear GAPDH (61, 168, and 241). To investigate the effects of extraction, cfDNA was analyzed directly using plasma and urine as well as in DNA extracts. To investigate the effects of storage, urine from 12 volunteers (per experiment) was immediately frozen or treated with EDTA and then frozen; stored with and without EDTA at room temperature and 4°C for 0 h, 1.5 h, 3 h and 5 h before freezing; and stored for 3 h with and without EDTA at room temperature and 4°C. Complete blood counts, urine pH, specific gravity and levels of hepatic/renal function biomarkers were obtained by an unspecified method.
Summary of Findings:
When plasma was amplified directly (i.e. DNA extraction was not performed), a minor peak at 178 bp was observed in the electropherograms; this peak was not observed when extraction was performed. Similarly, a peak at 62 bp was observed in urine specimens that were amplified directly (i.e. extraction was not performed); this peak was not observed when urine underwent DNA extraction. The peaks also differed between plasma and urine. Without ligation and amplification, cfDNA was not observable by gel electrophoresis regardless of whether DNA extraction was performed. When ligation and amplification were performed, a cfDNA smear was visible from extracted specimens but lacked shorter (<100 bp) and longer (>2,000 bp) fragments, which were visible among specimens that did not undergo extraction.
cfDNA concentration was comparable in urine when frozen at -80°C immediately or after the addition of EDTA; when stored at 4°C for 1.5, 3 or 5 h prior to the addition of EDTA; and when stored at 4°C for 3 h with and without EDTA. However, cfDNA yield was affected when urine was stored at room temperature for 1.5, 3 or 5 h before EDTA was added compared to 0 h (P<0.001), when stored at room temperature for 3 h compared to specimens without EDTA (P<0.001), and when stored without EDTA for 3 h at room temperature compared to matched controls stored at 4°C (P=0.003). The authors state that cfDNA fragment size was significantly affected by storage at 4°C or room temperature and by the addition of EDTA; consequently, they recommend adding EDTA immediately after urine collection, two rounds of centrifugation at 4°C, and then immediately freezing the supernatant at -80°C (data not shown). The mean copy numbers of long, medium and short amplicons of mitochondrial ND1 (94.84, 288.11, and 1084.98 copies/μL, respectively) were much higher than those of nuclear GAPDH (4.46, 15.53, and 6.32 copies/μL, respectively) in urine. The authors highlight that the copy numbers of ND1 decrease with increasing fragment size while GAPDH copies remain more consistent, with the highest numbers of ND1 observed for the medium length amplicon. The authors state there were no associations between GAPDH copy number and clinical parameters (blood cell counts, urinalysis results, and levels of hepatic/renal function biomarkers) in an initial analysis. However, the quantification cycle values (Cq) of the short amplicon of ND1 were correlated with lymphocyte percentage (Pearson r = -0.391, P = 0.002), neutrophil percentage (Pearson r =-0.344, P = 0.007), and the absolute value of lymphocyte counts (Spearman ρ = 0.364, P = 0.004). The copy number of the ND1 57 bp amplicon was correlated with pH (Spearman ρ = 0.258, P = 0.047), neutrophil percentage (Spearman ρ = 0.352, P = 0.006), urine specific gravity (Spearman ρ = -0.256, P = 0.048), absolute value lymphocyte counts (Spearman ρ = -0.397, P = 0.002), lymphocyte percentage (Spearman ρ = -0.410, P = 0.001). Additionally the copies of ND1 that were 57 -167 bp (ND1-S) were correlated with neutrophil percentage (Spearman ρ = 0.397, P = 0.002), lymphocyte percentage (Spearman ρ = -0.345, P = 0.001) and absolute lymphocyte count (Spearman ρ = -0.388, P = 0.002), and copies of ND1 between 167 and 240 bp (ND1-M) were correlated with absolute lymphocyte count (Spearman ρ = -0.306, P = 0.017). The ND1-S to ND1-M ratio was correlated with percentage neutrophils (Spearman ρ = -0.368, P = 0.004) and lymphocytes (Spearman ρ = 0.321, P = 0.012).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Electrophoresis Cell count/volume Hematology/ auto analyzer Small molecule pH DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation EDTA
None (fresh)
Refrigeration
Biospecimen Acquisition Biospecimen location Plasma
Urine
Storage Storage temperature 4°C
Room temperature
Analyte Extraction and Purification Analyte isolation method None
Extracted
Storage Storage conditions Frozen with EDTA
Directly frozen
Storage Storage duration 0 h
1.5 h
3 h
5 h