Abstract Introduction: Urine liquid biopsy is emerging as a non-invasive approach for disease monitoring and early detection. Transrenal DNA, cell-free DNA that passes from plasma through the kidney into urine, contains promising biomarkers that may expand the applications of urine liquid biopsy beyond urological malignancies. Due to the size restriction of the renal barrier, transrenal DNA is shorter than urological cfDNA (50 bp). However, the analysis of urine cfDNA is challenging as it is prone to rapid degradation. Furthermore, isolating short DNA fragments from the complex urine matrix demands optimized extraction protocols. The presented study addressed key technical challenges in urine liquid biopsy, presenting optimized workflows for stabilization, isolation, and analysis of urine cfDNA to realize the full potential of transrenal DNA research. Methods: Urine was collected from apparently healthy, consented individuals and either stabilized or left unstabilized. Urine samples were spiked with a DNA ladder with defined size fragments ranging from 10 to 300 bp. DNA was either isolated from urine on the day of collection, stabilization and spike-in, or after urine storage. DNA spike-in was also performed in phosphate-buffered saline. DNA was isolated either manually or automated with several commercially available cfDNA isolation technologies. The isolated cfDNA was analyzed by capillary gel electrophoresis. Results: All isolation kits tested were suitable for isolation of cfDNA down to 50 bp. Smaller DNA fragments down to 35 bp could be isolated with the miRNA protocol of the manual QIAamp® Circulating Nucleic Acid Kit as well as with automated isolation using the QIAsymphony® DSP Circulating DNA Kit and the EZ1 Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5329.
Mancarella-Langer et al. (Fri,) studied this question.