Simultaneous recovery of RNA and DNA from soils and sediments
Simultaneous recovery of RNA and DNA from soils and sediments
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DOI：
10.1128/aem.67.10.4495-4503.2001
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发表时间：
2001-10-01
影响因子：
4.4
通讯作者：
Zhou, JH
中科院分区：
文献类型：
--
作者：
Hurt, RA;Qiu, XY;Zhou, JH
从环境样品中回收mRNA用于测量原位代谢活性是一个重大挑战。开发了一种稳健、简单、快速和有效的方法，用于通过调整我们先前的基于研磨的细胞裂解方法从不同组成的土壤中同时回收RNA和DNA（Zhou等人，应用环境。Microbiol. 62：316-322，1996）用于DNA提取。其中一个关键的区别是，样品是在变性溶液中在低于0 ℃的温度下研磨，以抑制核酸酶活性。评价了两种不同的方法用于从DNA中分离RNA。在检测的RNA纯化方法中，阴离子交换树脂在RNA完整性、产率和纯度方面给出了最好的结果。利用优化后的方法，从19个不同组成的土壤和水系沉积物样品中同时回收了完整的RNA和高分子量DNA。这些样本的RNA产量范围从1.4到56微克土壤（-1）干重），而DNA产量范围从23到435微克（-1）。此外，对相同土壤样品的研究表明，DNA产量平均比我们以前的方法高40%，比商业珠磨方法高68%。对于大多数样品，回收的DNA和RNA具有足够的纯度用于核酸酶消化、微阵列杂交和PCR或逆转录-PCR扩增。
Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degreesC to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 mug g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 mug g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.