DNA extraction from long-term stored urine

Marc Hilhorst, Ruud Theunissen, Henk van Rie, Pieter van Paassen, Jan Willem Cohen Tervaert*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

17 Citations (Web of Science)

Abstract

Background: Traditionally, for DNA analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly, this option is often not considered. To complete our DNA database in patients with ANCA-associated vasculitis, we aimed to extract DNA from stored urine. Methods: Urine was stored at the time of kidney biopsy from patients included in our regional kidney biopsy database, who had given informed consent for further study. Urine was subsequently filtered, dialyzed, concentrated and freeze dried and finallyresolubilized and centrifuged. DNA was extracted using the high pure PCR template preparation kit (Roche Diagnostics). Next, concentration and purity were determined by Nanodrop analysis and by Quant-iT analysis. Results: One hundred and eighty-one patients with ANCA-associated vasculitis were included. Of 114 patients (63%), DNA was available. From 53 of the remaining 67 patients, stored urine was available. Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7 ng/mu L (range 33.2-529) with a mean purity ratio of 1.81 (lambda 260/280). Conclusion: DNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4 degrees C or lower temperatures results in significant degradation of the DNA, making recovery of DNA more difficult with longer periods of storage. In the current study, we demonstrated that DNA could be retrieved from subsequently filtered, dialyzed, concentrated and freeze dried urine that was stored at room temperature. In addition, we demonstrated tthat this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.
Original languageEnglish
Article number238
JournalBmc Nephrology
Volume14
DOIs
Publication statusPublished - 30 Oct 2013

Keywords

  • DNA
  • Urine
  • Vasculitis

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