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Implications of storing urinary DNA from different populations for molecular analyses.

Cannas A, Kalunga G, Green C, Calvo L, Katemangwe P, Reither K, Perkins MD, Maboko L, Hoelscher M, Talbot EA, Mwaba P, Zumla AI, Girardi E, Huggett JF, TB trDNA consorti - PLoS ONE (2009)

Bottom Line: The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.Site-specific differences in urine composition significantly affect the stability of DNA during storage.Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Infectious Diseases L. Spallanzani, IRCCS, Roma, Italy.

ABSTRACT

Background: Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.

Methodology: Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4 degrees C, -20 degrees C & -80 degrees C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.

Conclusion: Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.

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Direct comparison of human DNA stability in samples stored at −20°C over 28 days.The Italian urines stored with 40 mM EDTA at −20 C show the same biphasic degradation of human DNA observed in all Zambian urines. This biphasic degradation of the Zambian urines occurs in both the presence and absence of EDTA. Geometric means +/−95% confidence intervals are plotted for each treatment.
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pone-0006985-g004: Direct comparison of human DNA stability in samples stored at −20°C over 28 days.The Italian urines stored with 40 mM EDTA at −20 C show the same biphasic degradation of human DNA observed in all Zambian urines. This biphasic degradation of the Zambian urines occurs in both the presence and absence of EDTA. Geometric means +/−95% confidence intervals are plotted for each treatment.

Mentions: The addition of 40 mM EDTA to the Italian samples prevented degradation for 28 days when the samples were stored at room temperature, 4°C and −80°C; with an average loss of only 1.6% compared to baseline (data not shown). However, for urines stored at −20°C the addition of 40 mM EDTA did not prevent a ∼94.7% loss of DNA by day 28 (figure 4). With the Zambian samples, the addition of 10 mM EDTA solution, reflecting many commercially available urine preservation kits [21], [22], [23], had no stabilising effect regardless of storage temperature (data not shown). At this site, an average of ∼98.9% of the baseline DNA quantity was lost by day 7, increasing to ∼99.3% by day 28.


Implications of storing urinary DNA from different populations for molecular analyses.

Cannas A, Kalunga G, Green C, Calvo L, Katemangwe P, Reither K, Perkins MD, Maboko L, Hoelscher M, Talbot EA, Mwaba P, Zumla AI, Girardi E, Huggett JF, TB trDNA consorti - PLoS ONE (2009)

Direct comparison of human DNA stability in samples stored at −20°C over 28 days.The Italian urines stored with 40 mM EDTA at −20 C show the same biphasic degradation of human DNA observed in all Zambian urines. This biphasic degradation of the Zambian urines occurs in both the presence and absence of EDTA. Geometric means +/−95% confidence intervals are plotted for each treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735781&req=5

pone-0006985-g004: Direct comparison of human DNA stability in samples stored at −20°C over 28 days.The Italian urines stored with 40 mM EDTA at −20 C show the same biphasic degradation of human DNA observed in all Zambian urines. This biphasic degradation of the Zambian urines occurs in both the presence and absence of EDTA. Geometric means +/−95% confidence intervals are plotted for each treatment.
Mentions: The addition of 40 mM EDTA to the Italian samples prevented degradation for 28 days when the samples were stored at room temperature, 4°C and −80°C; with an average loss of only 1.6% compared to baseline (data not shown). However, for urines stored at −20°C the addition of 40 mM EDTA did not prevent a ∼94.7% loss of DNA by day 28 (figure 4). With the Zambian samples, the addition of 10 mM EDTA solution, reflecting many commercially available urine preservation kits [21], [22], [23], had no stabilising effect regardless of storage temperature (data not shown). At this site, an average of ∼98.9% of the baseline DNA quantity was lost by day 7, increasing to ∼99.3% by day 28.

Bottom Line: The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.Site-specific differences in urine composition significantly affect the stability of DNA during storage.Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Infectious Diseases L. Spallanzani, IRCCS, Roma, Italy.

ABSTRACT

Background: Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.

Methodology: Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4 degrees C, -20 degrees C & -80 degrees C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.

Conclusion: Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.

Show MeSH
Related in: MedlinePlus