Dataset Information

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

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.

SUBMITTER: Cannas A

PROVIDER: S-EPMC2735781 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Cannas Angela A Kalunga Glendah G Green Clare C Calvo Ludovica L Katemangwe Patrick P Reither Klaus K Perkins Mark D MD Maboko Leonard L Hoelscher Michael M Talbot Elizabeth A EA Mwaba Peter P Zumla Alimuddin I AI Girardi Enrico E Huggett Jim F JF

PloS one 20090910 9

<h4>Background</h4>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.<h4>Methodology</h4>Urine from 40 (20 male) healthy volunteers from two populations, Ital ...[more]

PMID: 19746164

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Project description:Research on insect microbiota has greatly expanded over the past decade, along with a growing appreciation of the microbial contributions to insect ecology and evolution. Many of these studies use DNA sequencing to characterize the diversity and composition of insect-associated microbial communities. The choice of strategies used for specimen collection, storage, and handling could introduce biases in molecular assessments of insect microbiota, but such potential influences have not been systematically evaluated. Likewise, although it is common practice to surface sterilize insects prior to DNA extraction, it is not known if this time-consuming step has any effect on microbial community analyses. To resolve these methodological unknowns, we conducted an experiment wherein replicate individual insects of four species were stored intact for two months using five different methods-freezing, ethanol, dimethyl sulfoxide (DMSO), cetrimonium bromide (CTAB), and room-temperature storage without preservative-and then subjected to whole-specimen 16S rRNA gene sequencing to assess whether the structure of the insect-associated bacterial communities was impacted by these different storage strategies. Overall, different insect species harbored markedly distinct bacterial communities, a pattern that was highly robust to the method used to store samples. Storage method had little to no effect on assessments of microbiota composition, and the magnitude of the effect differed among the insect species examined. No single method emerged as "best," i.e., one consistently having the highest similarity in community structure to control specimens, which were not stored prior to homogenization and DNA sequencing. We also found that surface sterilization did not change bacterial community structure as compared to unsterilized insects, presumably due to the vastly greater microbial biomass inside the insect body relative to its surface. We therefore recommend that researchers can use any of the methods tested here, and base their choice according to practical considerations such as prior use, cost, and availability in the field, although we still advise that all samples within a study be handled in an identical manner when possible. We also suggest that, in large-scale molecular studies of hundreds of insect specimens, surface sterilization may not be worth the time and effort involved. This information should help researchers design sampling strategies and will facilitate cross-comparisons and meta-analyses of microbial community data obtained from insect specimens preserved in different ways.

Dataset Information

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

Background

Methodology

Conclusion

Publications

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

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