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DNA extraction from long-term stored urine.

Hilhorst M, Theunissen R, van Rie H, van Paassen P, Tervaert JW - BMC Nephrol (2013)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical & Experimental Immunology, Maastricht University, P,O, Box 5800, 6202 AZ Maastricht, The Netherlands. jw.cohentervaert@maastrichtuniversity.nl.

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).

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 that this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.

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DNA from urine and DNA from buffycoat from three patients. The agarose gel shows CTLA-4 +49 polymorphism signals from these three patients. Patient 1 with buffycoat DNA on 1 and urine DNA on 2 (GG); patient 2 with buffycoat DNA on 3 and urine DNA on 4 (AA); patient 3 with buffycoat DNA on 5 and urine DNA on 6 (AA). DNA from urine and DNA from buffycoat yield similar signals in varying magnitude.
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Figure 2: DNA from urine and DNA from buffycoat from three patients. The agarose gel shows CTLA-4 +49 polymorphism signals from these three patients. Patient 1 with buffycoat DNA on 1 and urine DNA on 2 (GG); patient 2 with buffycoat DNA on 3 and urine DNA on 4 (AA); patient 3 with buffycoat DNA on 5 and urine DNA on 6 (AA). DNA from urine and DNA from buffycoat yield similar signals in varying magnitude.

Mentions: Freeze dried urine from these patients had been stored at room temperature for an average time of 16 years (range 6–28). 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) 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 40 ng/μL on the Nanodrop® 2100 and an average concentration of dsDNA of 23.35 ng/μL by picogreen analysis. Polymorphisms in several genes (CTLA-4, PD1) could be determined in 38 (82.6%) of these samples[5] (Figure 1). 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 (Figure 2).


DNA extraction from long-term stored urine.

Hilhorst M, Theunissen R, van Rie H, van Paassen P, Tervaert JW - BMC Nephrol (2013)

DNA from urine and DNA from buffycoat from three patients. The agarose gel shows CTLA-4 +49 polymorphism signals from these three patients. Patient 1 with buffycoat DNA on 1 and urine DNA on 2 (GG); patient 2 with buffycoat DNA on 3 and urine DNA on 4 (AA); patient 3 with buffycoat DNA on 5 and urine DNA on 6 (AA). DNA from urine and DNA from buffycoat yield similar signals in varying magnitude.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231352&req=5

Figure 2: DNA from urine and DNA from buffycoat from three patients. The agarose gel shows CTLA-4 +49 polymorphism signals from these three patients. Patient 1 with buffycoat DNA on 1 and urine DNA on 2 (GG); patient 2 with buffycoat DNA on 3 and urine DNA on 4 (AA); patient 3 with buffycoat DNA on 5 and urine DNA on 6 (AA). DNA from urine and DNA from buffycoat yield similar signals in varying magnitude.
Mentions: Freeze dried urine from these patients had been stored at room temperature for an average time of 16 years (range 6–28). 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) 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 40 ng/μL on the Nanodrop® 2100 and an average concentration of dsDNA of 23.35 ng/μL by picogreen analysis. Polymorphisms in several genes (CTLA-4, PD1) could be determined in 38 (82.6%) of these samples[5] (Figure 1). 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 (Figure 2).

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical & Experimental Immunology, Maastricht University, P,O, Box 5800, 6202 AZ Maastricht, The Netherlands. jw.cohentervaert@maastrichtuniversity.nl.

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).

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 that this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.

Show MeSH
Related in: MedlinePlus