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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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Related in: MedlinePlus

Comparison of DNA measurement using Pico green and qPCR.The stability of human and total DNA in urine stored at −20 C with 40 mM EDTA and without EDTA shown the same trends (A). Geometric means +/−95% confidence intervals are plotted for each treatment. Measurement with both human qPCR targets correlate with the direct measurement of total nucleic acids using pico-green (B). QPCR using these targets is an appropriate surrogate measure for total DNA and has a greater dynamic range than pico-green for which the lower threshold for accurate measurements is approximately 10 pg/µl.
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pone-0006985-g006: Comparison of DNA measurement using Pico green and qPCR.The stability of human and total DNA in urine stored at −20 C with 40 mM EDTA and without EDTA shown the same trends (A). Geometric means +/−95% confidence intervals are plotted for each treatment. Measurement with both human qPCR targets correlate with the direct measurement of total nucleic acids using pico-green (B). QPCR using these targets is an appropriate surrogate measure for total DNA and has a greater dynamic range than pico-green for which the lower threshold for accurate measurements is approximately 10 pg/µl.

Mentions: To confirm this finding the Italian analysis was repeated on a second group of volunteers by storing 20 healthy urine samples for seven days and assessed using the ALU J and TLR2 qPCR assays as well as by measuring total DNA using pico green. When urine was stored at −20 with 40 mM EDTA the degradation in the second group was identical to the first group (figure 5). Furthermore assessment of these samples using the Pico Green DNA measurement method confirmed the qPCR findings at −20°C with 40 mM EDTA (figure 6A) and supported the use of qPCR human targets as a surrogate measurement of total urinary DNA (figure 6B). We found that ∼100,000 copies of ALU J and ∼100 copies of TLR2 are equivalent to ∼47 pg and ∼65 pg of total DNA respectively.


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)

Comparison of DNA measurement using Pico green and qPCR.The stability of human and total DNA in urine stored at −20 C with 40 mM EDTA and without EDTA shown the same trends (A). Geometric means +/−95% confidence intervals are plotted for each treatment. Measurement with both human qPCR targets correlate with the direct measurement of total nucleic acids using pico-green (B). QPCR using these targets is an appropriate surrogate measure for total DNA and has a greater dynamic range than pico-green for which the lower threshold for accurate measurements is approximately 10 pg/µl.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006985-g006: Comparison of DNA measurement using Pico green and qPCR.The stability of human and total DNA in urine stored at −20 C with 40 mM EDTA and without EDTA shown the same trends (A). Geometric means +/−95% confidence intervals are plotted for each treatment. Measurement with both human qPCR targets correlate with the direct measurement of total nucleic acids using pico-green (B). QPCR using these targets is an appropriate surrogate measure for total DNA and has a greater dynamic range than pico-green for which the lower threshold for accurate measurements is approximately 10 pg/µl.
Mentions: To confirm this finding the Italian analysis was repeated on a second group of volunteers by storing 20 healthy urine samples for seven days and assessed using the ALU J and TLR2 qPCR assays as well as by measuring total DNA using pico green. When urine was stored at −20 with 40 mM EDTA the degradation in the second group was identical to the first group (figure 5). Furthermore assessment of these samples using the Pico Green DNA measurement method confirmed the qPCR findings at −20°C with 40 mM EDTA (figure 6A) and supported the use of qPCR human targets as a surrogate measurement of total urinary DNA (figure 6B). We found that ∼100,000 copies of ALU J and ∼100 copies of TLR2 are equivalent to ∼47 pg and ∼65 pg of total DNA respectively.

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