A protocol for urine collection and storage prior to DNA methylation analysis

• DOI: 10.1371/journal.pone.0200906
• Publication Date: 2018-08-24T13:26:28Z
• Authors: J. Bosschieter; S. Bach; I. V. Bijnsdorp; L. I. Segerink; W. F. Rurup; A. P. van Splunter; I. Bahce; P. W. Novianti; G. Kazemier; R. J. A. van Moor...; R. D. M. Steenbergen; J. A. Nieuwenhuijzen
• Keywords: 0301 basic medicine; Lung Neoplasms; Science; Preservation, Biological; Pilot Projects; Polymerase Chain Reaction; Specimen Handling; 03 medical and health sciences; SDG 3 - Good Health and Well-being; Carcinoma, Non-Small-Cell Lung; Biomarkers, Tumor; Humans; Edetic Acid; Urine Specimen Collection; Q; R; Liquid Biopsy; DNA; DNA, Neoplasm; DNA Methylation; 6. Clean water; 3. Good health; Urinary Bladder Neoplasms; Medicine; Research Article

Background Urine poses an attractive non-invasive means for obtaining liquid biopsies oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of DNA, particular hypermethylated (hmDNA), are yet to be determined. Aim To determine the most urine adequate hmDNA analysis. Methods yield use methylation was determined by quantitative specific PCR (qMSP) targeting ACTB RASSF1A genes bisulfite modified DNA. First, (ACTB qMSP) pilot study samples healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) time periods (1, 2, 7, 28 days). Next, levels (RASSF1A stored patients suffering from bladder cancer (n = 10) or non-small cell lung (NSCLC; n were measured day 0 7 upon with without addition 40mM EDTA and/or 20 μl/ml Penicillin Streptomycin (PenStrep) RT 4°C. Results In study, only maintained preserving agents. 4°C period days more, either agent yielded slightly better When -20 °C -80 up days, retained irrespective NSCLC loss significantly less if added compared no agents (p<0.001). Addition PenStrep did not affect (p>0.99). Upon difference found after (p 0.18). The methylated (RASSF1A) strongly correlated unmethylated (ACTB) cases, except when values became inaccurate. Conclusions offers inexpensive seven allowing reliable avoid bacterial overgrowth can negatively affecting preservation.