Preservation of nucleic acids by freeze-drying for next generation sequencing analyses of soil microbial communities
Preservation of nucleic acids by freeze-drying for next generation sequencing analyses of soil microbial communities
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DOI：
10.1093/jpe/rtw042
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发表时间：
2017-02-01
影响因子：
2.7
通讯作者：
Wubet, Tesfaye
中科院分区：
文献类型：
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作者：
Weissbecker, Christina;Buscot, Francois;Wubet, Tesfaye
目的土壤样品保存是土壤微生物群落分子研究中具有挑战性的一个方面。对于专门的样本存储设备、化学品和核酸提取的标准化协议的需求，往往需要在家庭实验室进行样本处理，而家庭实验室可以是远离采样点的大陆。标准采样程序，特别是在处理RNA时，包括立即将土壤快速冻结在液氮中，并在-80摄氏度下储存，直到进一步处理。对于这些情况，组织一条可靠的冷却链在大陆之间运输数百个土壤样本是非常昂贵的，如果可能的话。本研究通过对土壤微生物群落的对比条形码分析，测试了冻干和随后在4℃或环境温度下短期储存的土壤样品的保存效果，并与-80℃冻结的土壤样品进行了比较。方法采集了德国中部的两个草原土壤样品，在生物多样性探索区海尼奇-顿进行了采集。样本被冷冻干燥或储存在零下80摄氏度作为对照。冷冻干燥的样品储存在4摄氏度或环境温度下。所考察的两种储存温度的储存时间分别为1天和7天。提取总DNA和RNA，对细菌和丛枝菌根(AM)真菌群落分别进行16S(V4-V5可变区)和18S(NS31-AM1片段)的扩增454焦磷酸测序。重要发现细菌群落在rDNA和rRNA水平上得到了充分的保存，尽管冻干样品的储存效果略有降低，α多样性指数略有下降。AM真菌群落的研究可以在rDNA和rRNA水平上不发生明显变化。我们的结果表明，适当的采样设计，然后立即冻干土壤样品，可以使土壤样品在短期内跨越大陆运输。
Aims Soil sample preservation is a challenging aspect in molecular studies on soil microbial communities. The demands for specialized sample storage equipment, chemicals and standardized protocols for nucleic acid extraction often require sample processing in a home laboratory that can be continents apart from sampling sites. Standard sampling procedures, especially when dealing with RNA, comprise immediate snap freezing of soils in liquid nitrogen and storage at - 80 degrees C until further processing. For these instances, organizing a reliable cooling chain to transport hundreds of soil samples between continents is very costly, if possible at all. In this study we tested the effect of soil sample preservation by freeze-drying with subsequent short-term storage at 4 degrees C or ambient temperatures compared to -80 degrees C freezing by comparative barcoding analyses of soil microbial communities.Methods Two grassland soil samples were collected in Central Germany in the Biodiversity Exploratory Hainich-Dun. Samples were freeze-dried or stored at -80 degrees C as controls. Freeze-dried samples were stored at 4 degrees C or ambient temperature. Investigated storage times for both storage temperatures were 1 and 7 days. Total DNA and RNA were extracted and bacterial and arbuscular mycorrhizal (AM) fungal communities were analyzed by amplicon 454 pyrosequencing of the 16S (V4-V5 variable region) and 18S (NS31-AM1 fragment) of ribosomal RNA (rRNA) marker genes, respectively.Important Findings Bacterial communities were sufficiently well preserved at the rDNA and rRNA level although storage effects showed as slightly decreased alpha diversity indices for the prolonged storage of freeze-dried samples for 7 days. AM fungal communities could be studied without significant changes at the rDNA and rRNA level. Our results suggest that proper sampling design followed by immediate freeze-drying of soil samples enables short-term transportation of soil samples across continents.