Pei Li1,2, Jun Ning1, Xipeng Luo2, Hongli Du3, Qing Zhang2, Ganlin Zhou2, Qiu Du1, Zhenyu Ou1, Long Wang1, Yu Wang2. 1. XiangYa Hospital of Central South University, Changsha, Hunan, China. 2. Hunan UPSBio, Inc., Hunan University National Science Park, Changsha, Hunan, China. 3. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong, China.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: 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. METHODS: Urine samples were collected from bladder cancerpatients 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. RESULTS: 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. CONCLUSION: 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.
Authors: Takeo Fujii; Afsaneh Barzi; Andrea Sartore-Bianchi; Andrea Cassingena; Giulia Siravegna; Daniel D Karp; Sarina A Piha-Paul; Vivek Subbiah; Apostolia M Tsimberidou; Helen J Huang; Silvio Veronese; Federica Di Nicolantonio; Sandeep Pingle; Cecile Rose T Vibat; Saege Hancock; David Berz; Vladislava O Melnikova; Mark G Erlander; Rajyalakshmi Luthra; E Scott Kopetz; Funda Meric-Bernstam; Salvatore Siena; Heinz-Josef Lenz; Alberto Bardelli; Filip Janku Journal: Clin Cancer Res Date: 2017-01-17 Impact factor: 12.531
Authors: Olga E Bryzgunova; Tatyana E Skvortsova; Elena V Kolesnikova; Andrey V Starikov; Elena Yu Rykova; Valentin V Vlassov; Pavel P Laktionov Journal: Ann N Y Acad Sci Date: 2006-09 Impact factor: 5.691
Authors: Tibor Szarvas; Ilona Kovalszky; Katalin Bedi; Attila Szendroi; Attila Majoros; Péter Riesz; Tibor Füle; Viktória László; András Kiss; Imre Romics Journal: Oncol Rep Date: 2007-08 Impact factor: 3.906
Authors: Vanessa García Moreira; Belen Prieto García; Jose M Baltar Martín; Francisco Ortega Suárez; Francisco V Alvarez Journal: Clin Chem Date: 2009-09-03 Impact factor: 8.327
Authors: Nancy B Y Tsui; Peiyong Jiang; Katherine C K Chow; Xiaoxi Su; Tak Y Leung; Hao Sun; K C Allen Chan; Rossa W K Chiu; Y M Dennis Lo Journal: PLoS One Date: 2012-10-31 Impact factor: 3.240
Authors: Sarah M Dermody; Chandan Bhambhani; Paul L Swiecicki; J Chad Brenner; Muneesh Tewari Journal: Front Genet Date: 2022-04-27 Impact factor: 4.772
Authors: Danny Laurent; Fiona Semple; Philip J Starkey Lewis; Elaine Rose; Holly A Black; Jennifer Coe; Stuart J Forbes; Mark J Arends; James W Dear; Timothy J Aitman Journal: BMC Med Genomics Date: 2020-04-06 Impact factor: 3.063
Authors: Zuzana Pös; Ondrej Pös; Jakub Styk; Angelika Mocova; Lucia Strieskova; Jaroslav Budis; Ludevit Kadasi; Jan Radvanszky; Tomas Szemes Journal: Int J Mol Sci Date: 2020-11-16 Impact factor: 5.923