Quantifying cell free DNA in urine: comparison between commercial kits, impact of gender and inter-individual variation.
Author(s): Streleckiene G, Reid HM, Arnold N, Bauerschlag D, Forster M
Publication: Biotechniques, 2018, Vol. 64, Page 225-230
PubMed ID: 29793362 PubMed Review Paper? No
Purpose of Paper
This paper investigated differences between patient gender and extraction method used on the levels and integrity of cell-free DNA (cfDNA) in urine from healthy volunteers.
Conclusion of Paper
Pooled urine from females had higher levels of cfDNA than pooled urine from males, regardless of cfDNA extraction method, but inter-individual variation in cfDNA levels was high. The yield of cfDNA was higher when extraction was with the Perkin Elmer NEXTprep-Mag Urine cfDNA Isolation Kit than the Norgen Urine Cell-Free Circulating DNA Purification kit, but the difference was only significant for urine from males. The electropherograms showed a clear peak at 224-232 nt when isolation was with the Perkin Elmer NEXTprep-Mag Urine cfDNA Isolation Kit, but the peak was smaller and less distinct when isolation was with the Norgen Urine Cell-Free Circulating DNA purification kit.
Studies
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Study Purpose
The purpose of this study was to determine differences between patient gender and extraction method used on the levels and integrity of cfDNA in urine from healthy volunteers. First morning urine was collected from 8 healthy volunteers (4 male and 4 female) between 8 and 9 AM on five different days. Urine was centrifuged at 200 x g for 10 min followed by 1800 x g for 10 min starting between 10 and 11 AM. Equal volumes of urine from all volunteers of each gender were pooled. cfDNA was isolated from the pooled urine and on day 5 from individual urine specimens using both the NEXTprep-Mag Urine cfDNA Isolation Kit and the Urine Cell-Free Circulating DNA Purification kit. The length and concentration of the DNA were determined using a TapeStation.
Summary of Findings:
Pooled urine from females had higher levels of cfDNA than pooled urine from males (P=0.003), regardless of cfDNA extraction method, but inter-individual variation in cfDNA levels was high. The yield of cfDNA was higher in both pools and in 7 of 8 individual specimens when extraction was with the Perkin Elmer NEXTprep-Mag Urine cfDNA Isolation Kit than the Norgen Urine Cell-Free Circulating DNA Purification kit, but the difference was only significant for urine from males (P=0.008). Further, the electropherograms showed a clear peak at 224-232 nt when isolation was with the Perkin Elmer NEXTprep-Mag Urine cfDNA Isolation Kit, but the peak was smaller and less distinct when isolation was with the Norgen Urine Cell-Free Circulating DNA purification kit.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method NEXTprep-Mag Urine cfDNA Isolation Kit
Urine Cell-Free Circulating DNA Purification Kit
Preaquisition Patient gender Female
Male