Abstract B015: Collection and purification of microgram amounts of cfDNA from urine for methylation analysis

Abstract Background: DNA methylation studies often require relatively large amounts of DNA, typically methods suggest hundreds of nanograms to a microgram per assay. While this is easy to obtain if genomic DNA (gDNA) is used, preforming similar studies on circulating cell free DNA (cfDNA) may be difficult. Plasma, especially that from normal individuals needed for comparison, may yield only 10 ng to 20 ng per ml. Thus, experimental designs involving multiple repeat analysis of the same specimen, or applying different complementary methylation methods to one specimen, may be limited by the amount of cfDNA in a blood sample. It is known that urine contains cfDNA, but reports on the amount vary from very low to being comparable to blood; the conflicting data may be due in part to the low yield some methods of isolation have reported to have when the DNA is small. Hypothesis: We postulated that a system which filtered hundreds of mls of urine and captured the DNA based on charge rather than absorption to silica, could capture adequate amounts of cfDNA from urine for repeat studies. Results: Initially, filtering large amounts of urine was problematic due to clogging. A system of stacked filters was devised. The method uses 200- 250 ml size syringes and 5 cm filter cartridges. It allows full voids, in fact up to 1 liter, to be filtered. The urine is first collected in a measuring cup containing preservatives, it is immediately filtered. This is simple enough to be done by a patient at home. The first of the stacked filters, collects urothelial and other cells that may be used to analyze gDNA; it also clarifies the urine sufficiently to pass through a second lower pore size filter modified to be highly positively charged. On average, a 150 ml void provides 2100 nanograms of cfDNA. The binding to the filter is sufficiently strong that DNases cannot bind or digest the DNA. Thus, the DNA is stable in the cartridge for four days and allows for shipping without special handling. The in-lab purification consists of separating the filters, then washing the cfDNA trapping filter, followed by elution of the DNA. It takes under 10 minutes and results in purified DNA that is suitable for downstream analysis. Conclusions: Microgram amounts of cfDNA can be easily extracted from urine to facilitate initial analyses sufficient for statistically robust studies and banking of specimens for future testing. Citation Format: Floyd Taub, Lindsey Ray, Charles Preston Neff, Elijah Barstis, Charles Henry, Kirsten Arnold. Collection and purification of microgram amounts of cfDNA from urine for methylation analysis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B015.