The guanidine thiocyanate-high EDTA method for total microbial RNA extraction from severely heavy metal-contaminated soils.
The guanidine thiocyanate-high EDTA method for total microbial RNA extraction from severely heavy metal-contaminated soils.
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硫氰酸胍-高EDTA法提取重金属严重污染土壤中微生物总RNA
DOI：
10.1111/1751-7915.13615
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发表时间：
2021-03
影响因子：
5.7
通讯作者：
Li X
中科院分区：
文献类型：
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作者：
Pei Y;Mamtimin T;Ji J;Khan A;Kakade A;Zhou T;Yu Z;Zain H;Yang W;Ling Z;Zhang W;Zhang Y;Li X
基于RNA的分子生物学分析作为揭示活性微生物及其功能基因的一种直接手段，近年来越来越受到环境研究者的关注。然而，从严重重金属污染的土壤中提取足够的高质量的微生物总RNA仍然是一个挑战。在本研究中，建立并优化了硫氰酸胍-高EDTA（GTHE）方法，用于从长期重金属污染的土壤中回收大量和高质量的RNA。由于土壤中微生物生物量低，我们结合多种强变性剂和强烈的机械裂解来破碎细胞以增加RNA产量。为了尽量减少RNA酶和重金属对RNA完整性的干扰，硫氰酸胍和EDTA的浓度分别从0.5 ml g−1土壤增加到0.625 ml g−1土壤和10 mM增加到100 mM。将优化后的GTHE方法应用于7种严重污染的土壤，RNA回收率为2.80 ~ 59.41 μg g−1土。使用非Cr（VI）（NT）和Cr（VI）处理（CT）样本的总微生物RNA进行分子分析。qRT-PCR结果表明，Cr（VI）处理后，两个测试基因chrA和yieF的表达分别上调4.12倍和62.43倍。从NT和CT样品中提取的微生物总RNA分别达到26.70 μg和30.75 μg，远高于构建元转录组文库所需的5 μg。此外，mRNA读段率超过86%，这表明构建的用于元转录组学分析的高质量文库。总之，GTHE方法对于研究污染生境中的微生物是有用的。本研究开发并优化了从严重重金属污染土壤中分离高质量微生物总RNA的GTHE方法。优化后的GTHE法提取的RNA可用于综合分子分析。此外，所确定的RNA提取影响因素及相应的解决策略为其他土壤微生物总RNA提取方法提供了重要参考。
Molecular analyses relying on RNA, as a direct way to unravel active microbes and their functional genes, have received increasing attention from environmental researchers recently. However, extracting sufficient and high‐quality total microbial RNA from seriously heavy metal‐contaminated soils is still a challenge. In this study, the guanidine thiocyanate‐high EDTA (GTHE) method was established and optimized for recovering high quantity and quality of RNA from long‐term heavy metal‐contaminated soils. Due to the low microbial biomass in the soils, we combined multiple strong denaturants and intense mechanical lysis to break cells for increasing RNA yields. To minimize RNAase and heavy metals interference on RNA integrity, the concentrations of guanidine thiocyanate and EDTA were increased from 0.5 to 0.625 ml g−1 soil and 10 to 100 mM, respectively. This optimized GTHE method was applied to seven severely contaminated soils, and the RNA recovery efficiencies were 2.80 ~ 59.41 μg g−1 soil. The total microbial RNA of non‐Cr(VI) (NT) and Cr(VI)‐treated (CT) samples was utilized for molecular analyses. The result of qRT‐PCR demonstrated that the expressions of two tested genes, chrA and yieF, were respectively upregulated 4.12‐ and 62.43‐fold after Cr(VI) treatment. The total microbial RNA extracted from NT and CT samples, respectively, reached to 26.70 μg and 30.75 μg, which were much higher than the required amount (5 μg) for metatranscriptomic library construction. Besides, ratios of mRNA read were more than 86%, which indicated the high‐quality libraries constructed for metatranscriptomic analysis. In summary, the GTHE method is useful to study microbes of contaminated habitats. This study developed and optimized the GTHE method for isolating high‐quality total microbial RNA from severely heavy metal‐contaminated soils. The yielded RNA by the optimized GTHE method can be used for comprehensive molecular analyses. Further, the identified RNA extraction influencing factors and the corresponding solving strategies provide an important reference to other soil total microbial RNA extraction methods.
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影响因子：
4.4
作者：
Hurt, RA;Qiu, XY;Zhou, JH
通讯作者：
Zhou, JH
影响因子：
4.4
作者：
Griffiths, RI;Whiteley, AS;Bailey, MJ
通讯作者：
Bailey, MJ
影响因子：
2.2
作者：
Daniell, T. J.;Davidson, J.;Roberts, D. M.
通讯作者：
Roberts, D. M.
影响因子：
5
作者：
Jun E;Oh J;Lee S;Jun HR;Seo EH;Jang JY;Kim SC
通讯作者：
Kim SC
宏基因组学和宏转录组学揭示酸性矿山排水中优势和稀有原核生物的生态作用
DOI：
10.1038/ismej.2014.212
发表时间：
2015-06
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
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