Implications of storing urinary DNA from different populations for molecular analyses.
Implications of storing urinary DNA from different populations for molecular analyses.
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DOI：
10.1371/journal.pone.0006985
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发表时间：
2009-09-10
期刊：
影响因子：
3.7
通讯作者：
TB trDNA consortium
中科院分区：
文献类型：
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作者：
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 consortium
利用尿液进行分子诊断已被确立用于许多性传播疾病，并越来越多地用于诊断肿瘤和其他传染病。在分析之前储存尿液，无论是由于家庭收集还是生物银行，越来越多地被提倡，但还没有出现最佳实践。在这里，我们检测了两个人群储存尿液中DNA的稳定性超过28天。来自意大利和赞比亚两个人群的40名（20名男性）健康志愿者的尿液在有和没有EDTA防腐剂溶液的情况下储存在四种不同温度（RT、4°C、−20°C和−80°C）下。在储存后第0、1、3、7和28天抽取尿液。通过定量实时PCR使用多拷贝（ALU J）和单拷贝（TLR 2）靶标测量人DNA含量。赞比亚和意大利的样本在零时含有相当的DNA量。一般而言，在储存期间观察到两种趋势：无降解，或从第0天至第7天快速降解，随后至第28天几乎没有进一步降解。无论储存条件如何，在赞比亚总是观察到双相降解，但在意大利仅观察到两次。尿液成分的位点特异性差异显著影响DNA在储存期间的稳定性。通过使用本文所述的策略类型来评估储存的尿液用于分子分析的质量，在这些样本用于分子预后监测、遗传分析和疾病诊断之前是至关重要的。
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. Urine 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. 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.