DNA extraction from long-term stored urine (2024)

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 258.7ng/μL (range 33.2-529) with a mean purity ratio of 1.81 (λ 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°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.

Genetic factors have been studied by analysis of DNA in many different diseases. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, the relevance of this analysis was shown in a genomewide association study [1]. Traditionally, 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 ANCA-associated glomerulonephritis.

The Limburg Renal Registry [3] was searched to identify all ANCA positive patients with pauci-immune necrotizing crescentic glomerulonephritis in order to perform DNA analysis. Patients from who no buffy coats and/or tissue was available but who had given consent for further study, were included in the current study. The local Medical Research Ethics Committee of the Maastricht University Medical Centre approved the study.

Exclusion criteria were concomitant renal diseases such as diabetic nephropathy, thin GBM glomerulopathy or anti-GBM glomerulonephritis.

At the time of renal disease, urine of patients was filtered by passing it through a paper filter. The urine was then dialyzed and concentrated in a Proflux M12® (Millipore, Billerica, MA, USA). Dialyzed and concentrated urine was subsequently snap frozen in a bath of liquid nitrogen and dried in a Beta 1–8 LD® freeze dryer (Christ, Osterode, Germany). Samples were subsequently stored at room temperature until use. For this purpose, 0.2 grams of freeze dried urine was resolubilized in 20mL of MilliQ and left it stirring overnight at 4°C. We next centrifuged the urine samples for 10minutes at 10000g. We extracted DNA by using the high pure PCR template preparation kit (Roche Molecular Diagnostics, Mannheim, Germany). Briefly, the supernatant was decanted and the sediment dissolved in 50μL proteinase K in 1mL of binding buffer (6M guaninidine-HCl, 10mM urea, 10mM Tris–HCl, 20% Triton X-100, pH4.4). After a ten minute incubation at 56°C, 100μL of iso-propanol was added and the solution was centrifuged through a filtertube containing glass fibers for 1minute at 8000g. The filtertube was subsequently centrifuged with 500μL of inhibitor removal buffer (5M guaninidine-HCl, 20mM Tris–HCl, 45% ethanol, pH6.6) for 1minute at 8000g and washed three times with wash buffer (20mM NaCl, 2mM Tris–HCl, 80% ethanol, pH7.5) for 1minute at 8000g. The DNA on the glass fibers was then eluted in 200μL of elution buffer by centrifuging for 1minute at 8000g and measured for concentration and purity on a NanoDrop® Spectrophotometer (Nanodrop Technologies, Wilmingtom, DE, USA). In addition, DNA was measured using the Quant-iT™ Picogreen® dsDNA assay. This is an ultrasensitive fluorescent nucleic acid staining for quantitating small amounts of double stranded DNA [4].

In the current study, 181 consecutive patients with ANCA-associated glomerulonephritis were included. Of 114 patients (63%) DNA was available. From 53 of the remaining 67 patients, 24hour freeze dried urine was available.

Freeze dried urine from these patients had been stored at room temperature for an average time of 16years (range 6–28). Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7ng/μL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280) as measured on a Nanodrop® 2100. Eleven samples were further diluted for picogreen analysis. These samples were found to contain DNA in an average concentration of 40ng/μL on the Nanodrop® 2100 and an average concentration of dsDNA of 23.35ng/μL by picogreen analysis. Polymorphisms in several genes (CTLA-4, PD1) could be determined in 38 (82.6%) of these samples [5] (Figure1). 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). Furthermore, Chi-square analysis proved gender to be of no significance (p = 0.8) yielding signals in 87.5% of males and in 54.5% of females. Finally, gel electrophoresis with PCR products from DNA from urine and from buffy coat of three patients showed similar bands in varying magnitude (Figure2).

DNA:

Deoxyribonucleic acid

ANCA:

Anti-neutrophil cytoplasmic antibodies

GBM:

Glomerular basem*nt membrane

PCR:

Polymerase chain reaction.

Authors and Affiliations

1. Clinical & Experimental Immunology, Maastricht University, P.O. Box 5800, 6202, AZ, Maastricht, The Netherlands

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

Corresponding author

Correspondence to Jan Willem Cohen Tervaert.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

MH, RT and HvR performed extraction of DNA and critical revision of the manuscript. MH and JWCT contributed to the concept of study, interpretation of data, draft of the manuscript and critical revision of the manuscript. PvP contributed to concept of study, interpretation of data and critical revision of the manuscript. All authors have given final approval of the manuscript to be submitted.

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions