Genotyping for DQA1 and PM loci in urine using PCR-based amplification: Effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing

Vu, Nicole; Chaturvedi, Arvind; Canfield, Dennis V.

doi:10.1016/s0379-0738(99)00034-1

Cited by 38 publications

(46 citation statements)

References 7 publications

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“…A similar reduction in the amount of human urinary DNA at baseline in males has been recorded for studies in USA [17], [27], Germany [18], [20], but was not observed in healthy individuals from Russia [28] or the Zambian individuals in this study.…”

Section: Discussionsupporting

confidence: 88%

“…Studies whose primary outcome was the measurement of bacterial DNA have concluded that storage at 4°C for up to 30 days with EDTA [15], or for 1 week without EDTA [16] did not affect molecular detection. Studies whose primary outcome was the recovery and analysis of human DNA have concluded that storing urine at room temperature with sodium azide over 30 days [17] or at −20°C with EDTA over 72 days [18], provides the best storage method. Furthermore, in other studies, useful human molecular analyses have been obtained from urine stored without preservative at −20°C for up to 25 years [19] and up to 7 years [20].…”

Section: Introductionmentioning

confidence: 99%

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Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

et al. 2009

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BackgroundMolecular 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.MethodologyUrine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4°C, −20°C & −80°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.ConclusionSite-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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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

et al. 2009

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“…Because of the small number of samples used in our study, the power is limited and negative results should be interpreted with caution. The pellet can be used for DNA-based analyses such as genotyping 35 or urinary metagenomics analyses. 28 Microparticles in the supernatant can be isolated for further analyses by ultracentrifugation at a later stage, after storage at -80°C.…”

Section: Urine Processing Methods 355 Discussionmentioning

confidence: 99%

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

Ammerlaan

Trezzi

Mathay

et al. 2014

Biopreservation and Biobanking

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Background: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. Methods: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. Results: The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. Conclusions: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.

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“…Furthermore, the literature differs concerning the appropriate storage conditions and duration for urine samples. Urine samples with sodium azide can be stored for 20 days at room temperature (Vu et al, 1999), whereas the storage time for those with EDTA is N72 days (Milde et al, 1999). van der Hel et al reported that urine stored at −20°C for 25 years without preservatives could still be used for molecular biology tests (van der Hel et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: A qualitative study

Wang

Chen²,

Nan³

et al. 2016

Gene

176

110

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Genotyping for DQA1 and PM loci in urine using PCR-based amplification: Effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing

Cited by 38 publications

References 7 publications

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: A qualitative study

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