New method to preserve the original proportion and integrity of urinary cell-free DNA

Due to high nuclease activity and complex contents in urine, urinary cell-free DNA (ucfDNA) was prone to degrade. So, we developed standardized urine collection tube (UCT) to prevent ucfDNA degradation and simultaneously maintain urinary cells in their original form during the sample collection process, ensuring stabilization of the original proportion and integrity of ucfDNA.

Urine samples were collected from bladder cancer patients and divided into 10-mL normal tubes and 10-mL UCTs, respectively, and kept at ambient temperature. Urine supernatant was separated by centrifuging, and ucfDNA was extracted. Then ucfDNA was quantified by quantitative real-time polymerase chain reaction. UcfDNA fragments distribution was analyzed by Agilent 2200, and the frequency of specific mutations of urinary system disease was detected by next-generation sequencing method.

Urine collected into UCTs showed no statistically significant changes in their original proportion and integrity of ucfDNA up to 7 days at ambient temperature and also ucfDNA fragments were maintained well. Conversely, urine collected into normal tubes was observed an obviously decline in their original proportion of ucfDNA and ucfDNA fragments changed greatly. The △% of allele fraction (AF) for specific genes of ucfDNA from UCTs was lower than from normal tubes by 3.7-fold.

Using UCTs, they can maximally keep the original proportion and integrity of ucfDNA and stabilize urinary cells and minimize the background noise caused by urinary cellular DNA releasing, it will be help to open the door of next-generation noninvasive liquid biopsy applications utilizing urine.