Abstract
BackgroundTraditionally, 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.MethodsUrine 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.ResultsOne 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).ConclusionDNA 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 tthat this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.
Highlights
For Deoxyribonucleic acid (DNA) analyses, DNA is recovered from buffy coats
Freeze dried urine from these patients had been stored at room temperature for an average time of 16 years
Linear regression showed that the amount of DNA extracted from the urine did not correlate with the amount of proteinuria at the time of urine storage (R2 = 0.02; p = 0.34) or the length of time that the urine was stored (R2 = 0.08; p = 0.55)
Summary
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. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, the relevance of this analysis was shown in a genomewide association study [1]. For these analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly [2], urine is generally not used for the purpose of DNA analysis. We postulate that it is possible to extract DNA from appropriately stored urine from patients with ANCAassociated glomerulonephritis
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