Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine

Publication date: Available online 20 August 2019Source: The Journal of Molecular DiagnosticsAuthor(s): Amy Oreskovic, Norman D. Brault, Nuttada Panpradist, James J. Lai, Barry R. LutzUrine cell-free DNA (cfDNA) is a valuable noninvasive biomarker for detecting cancer mutation, diagnosing infectious disease (eg, tuberculosis), monitoring organ transplantation, and prenatal screening. Conventional silica DNA extraction does not efficiently capture urine cfDNA, which is dilute (ng/mL) and highly fragmented (30 to 100 nt). The clinical sensitivity of urine cfDNA detection increases with decreasing target length, motivating use of sample preparation methods designed for short fragments. We compared the analytical performance of two published protocols (Wizard/GuSCN and Q Sepharose), three commercial kits (Norgen, QIAamp, and MagMAX), and an in-house sequence-specific hybridization capture technique. Dependence on fragment length (25 to 150 nt), performance at low concentrations (10 copies/mL), tolerance to variable urine conditions, and susceptibility to PCR inhibition was characterized. Hybridization capture and Q Sepharose performed best overall (60% to 90% recovery), although Q Sepharose had reduced recovery (<10%) of the shortest 25 nt fragment. Wizard/GuSCN recovery was dependent on pH and background DNA concentration and was limited to <35% even under optimal conditions. The Norgen kit suffered from consistent PCR inhibition but had high recovery of short fragments....
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research