Unlocking transrenal DNA: New methods for stabilizing and isolating small cfDNA in urine

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&2® ccfDNA Kit. With the integration of new nucleic acid binding beads into the automated isolation with the QIAsymphony DSP Circulating DNA Kit, isolation of cfDNA down to 20 bp was possible. To test the need for urine stabilization for efficient isolation of small cfDNA from urine, the spike-in DNA was isolated from unstabilized urine as well as urine stabilized with the PAXgene® Urine Liquid Biopsy Set. For unstabilized samples, the ladder fragments could not be detected even when isolated within hours of urine collection, indicating rapid degradation of the ladder DNA. For stabilized urine samples, the DNA ladder spike-in could be detected even after days of storage. Conclusion: This study emphasizes the critical role for urine stabilization for efficient isolation of a wide range of cfDNA sizes. The isolation technology defines the cfDNA size range available for analysis and should be chosen based on the cfDNA population of interest. The PAXgene Urine Liquid Biopsy Set, combined with manual or automated isolation technologies, enables recovery of small cfDNA fragments and hence, transrenal DNA research.