Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils

Töwe, Stefanie; Wallisch, Stefanie; Bannert, Andrea; Fischer, Doreen; Hai, Brigitte; Haesler, Felix; Kleineidam, Kristina; Schloter, Michael

doi:10.1016/j.mimet.2010.12.028

Cited by 69 publications

(34 citation statements)

References 40 publications

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“…Our results demonstrate that the 9 different protocols actually result in a distinct phylogenetic composition of the nucleic acids recovered. This substantiates previous findings that were based on low-resolution fingerprint analyses (24,26,27).…”

Section: Factors Affecting Diversity Analysessupporting

confidence: 92%

“…Indirect methods typically yield longer nucleic acid fragments which are useful for metagenomic studies (10,15) but often result in a significantly lower yield and diversity of nucleic acid molecules (10,16). Existing protocols for the direct extraction of DNA and RNA from soils (13,(17)(18)(19)(20)(21)(22)(23)(24)(25) have partly been evaluated (19,21,24,(26)(27)(28)(29)(30), but only a few studies have compared extraction efficiencies of methods recovering both DNA and RNA (21,24). In particular, these methods have so far not been compared with extraction protocols optimized for either DNA or RNA alone.…”

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confidence: 99%

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Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

Wüst

Nacke

Kaiser

et al. 2016

Appl Environ Microbiol

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dModern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actinobacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms. Soil is one of the most complex and diverse microbial habitats, with 1 g containing up to 10 10 cells and 10 4 bacterial species (1, 2). While DNA sequences provide information about the total microbial community, RNA can be used to analyze the fraction of microorganisms that has the capacity to actively synthesize proteins (3-5). The ratio of bacterial 16S rRNA transcripts to 16S rRNA gene copies has been used as an indicator of the potential specific activity since it reflects the cellular ribosome content (6, 7). However, nucleic acid extraction from soils is affected by insufficient cell lysis or losses during extraction, variable reproducibility, and various effects of soil pH, clay content, and organic carbon content (8-13), and coextracted organic compounds can inhibit DNA polymerase and reverse transcriptase employed in downstream molecular analyses (14).Direct nucleic acid extraction methods are based on the lysis of cells inside the soil matrix, whereas indirect methods commence with the isolation of bacterial cells from soil prior to nucleic acid extraction (8). Indirect methods typically yield longer nucleic acid fragments which are useful for metagenomic studies (10, 15) but often result in a significantly lower yield and diversity of nucleic acid molecules (10, 16). Existing protocols for the direct extraction of DNA and RNA from soils (13, 17-25) have partly been evaluated (19,21,24,(26)(27)(28)(29)(30)), but only a few studies have compared extraction efficiencies of methods recovering both DNA and RNA (21, 24). In particular, these methods have so far not been compared with extraction protocols optimized for either DNA or RNA alone. Information about the effects of different extraction methods on the phylogenetic analysis is scant (31, 32). As a result, ...

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Section: Factors Affecting Diversity Analysessupporting

confidence: 92%

mentioning

confidence: 99%

Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

Wüst

Nacke

Kaiser

et al. 2016

Appl Environ Microbiol

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“…One gram of macerated nodules (2× 0.5 g) was extracted according to Töwe et al (2011). The quality and quantity of the DNA extracts were evaluated in 1% agarose gels and using the PicoGreen dsDNA quantification assay (Invitrogen, USA), respectively.…”

Section: Methodsmentioning

confidence: 99%

Cowpea Nodules Harbor Non-rhizobial Bacterial Communities that Are Shaped by Soil Type Rather than Plant Genotype

et al. 2017

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Many studies have been pointing to a high diversity of bacteria associated to legume root nodules. Even though most of these bacteria do not form nodules with legumes themselves, it was shown that they might enter infection threads when co-inoculated with rhizobial strains. The aim of this work was to describe the diversity of bacterial communities associated with cowpea (Vigna unguiculata L. Walp) root nodules using 16S rRNA gene amplicon sequencing, regarding the factors plant genotype and soil type. As expected, Bradyrhizobium was the most abundant genus of the detected genera. Furthermore, we found a high bacterial diversity associated to cowpea nodules; OTUs related to the genera Enterobacter, Chryseobacterium, Sphingobacterium, and unclassified Enterobacteriacea were the most abundant. The presence of these groups was significantly influenced by the soil type and, to a lesser extent, plant genotype. Interestingly, OTUs assigned to Chryseobacterium were highly abundant, particularly in samples obtained from an Ultisol soil. We confirmed their presence in root nodules and assessed their diversity using a target isolation approach. Though their functional role still needs to be addressed, we postulate that Chryseobacterium strains might help cowpea plant to cope with salt stress in semi-arid regions.

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“…DNA was extracted from 0.3 g soil via phenolchloroform-based extraction described by Töwe et al (2011), using a Precellys24 Instrument (PeqLab, Erlangen, Germany). Quality and quantity of the nucleic acids were checked using a spectrophotometer (Nanodrop, PeqLab, Erlangen, Germany) and gel electrophoresis.…”

Section: Dna Extraction Pcr and 454 Pyro-sequencingmentioning

confidence: 99%

Functional land-use change effects on soil fungal communities in Chilean temperate rainforests

Marín¹,

Godoy

Valenzuela

et al. 2017

J. Soil Sci. Plant Nutr.

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By reducing soil organic matter and litter input, land-use changes are predicted to decrease total soil fungal diversity, but at functional levels this have been poorly studied. It is expected, though, that increasing disturbance decreases saprotrophic and mycorrhizal fungi biodiversity. This study aimed to determine the effects of land-use changes on the phylogenetic and functional diversity of soil fungi in the Southern Andes. We assessed the fungal communities of Andosol topsoil at 1 cm and 10 cm soil depth. The soil samples were obtained from a gradient of anthropogenic disturbance; specifically, plots were located within pristine forest, overstory-managed, and clearcut conditions. We used a cultivation-independent molecular barcoding approach to assess fungal diversity and identify 1,173 OTUs from which 401 were assigned to a functional guild. While we found higher phylogenetic richness in clear-cut conditions, these soils had higher relative abundances of plant pathogen fungi and lower relative abundances of saprotrophic and ectomycorrhizal fungi compared to the other treatments. The opposite pattern was found in pristine forest. Thus, fungal species richness itself does not seem to reflect ecosystem health. Interestingly though, the lower phylogenetic diversity found in pristine forest was compensated by a higher diversity of fungi involved in nutrient cycling.

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Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils

Cited by 69 publications

References 40 publications

Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed

Cowpea Nodules Harbor Non-rhizobial Bacterial Communities that Are Shaped by Soil Type Rather than Plant Genotype

Functional land-use change effects on soil fungal communities in Chilean temperate rainforests

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