title: DNA extraction from long-term stored urine.
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 finally resolubilized 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/μL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280).
  
  
  CONCLUSIONS
  DNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4 °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 that this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.
authors.forename: Marc
authors.initials: M
authors.lastname: Hilhorst
authors.forename: Ruud
authors.initials: R
authors.lastname: Theunissen
authors.forename: Henk
authors.initials: H
authors.lastname: van Rie
authors.forename: Pieter
authors.initials: P
authors.lastname: van Paassen
authors.affiliation: Clinical & Experimental Immunology, Maastricht University, P,O, Box 5800, 6202 AZ Maastricht, The Netherlands. jw.cohentervaert@maastrichtuniversity.nl.
authors.forename: Jan Willem Cohen
authors.initials: JW
authors.lastname: Tervaert
mesh_terms: D056648:Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis / Q000453:epidemiology* / Q000652:urine*; D004247:DNA / Q000235:genetics* / Q000652:urine*; D004356:Drug Storage; D020022:Genetic Predisposition to Disease / Q000453:epidemiology / Q000235:genetics; D006801:Humans; D009426:Netherlands / Q000453:epidemiology; D016133:Polymerase Chain Reaction / Q000706:statistics & numerical data*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015995:Prevalence; D013048:Specimen Handling / Q000379:methods* / Q000706:statistics & numerical data*
chemical_list: D004247:DNA