A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

Lever, Mark A.; Torti, A.; Eickenbusch, P.; Michaud, Alexander B.; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

doi:10.3389/fmicb.2015.00476

Cited by 271 publications

(340 citation statements)

References 79 publications

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“…Libraries were pooled and sequenced over two lanes of a HiSeq 2500 (Illumina), employing 100 bp paired-end reads with SBS v3 reagents. Copy numbers of archaeal and bacterial 16S rRNA genes in DNA were determined by quantitative PCR on an Mx3005P instrument (Agilent) by using the primer combinations Bac908F_mod/Bac1075R for bacteria and 915Fmod/1059R for Archaea 61 .…”

Section: Methodsmentioning

confidence: 99%

Altered carbon turnover processes and microbiomes in soils under long-term extremely high CO2 exposure

et al. 2016

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* There is only limited understanding of the impact of high p(CO 2 ) on soil biomes. We have studied a floodplain wetland where long-term emanations of temperate volcanic CO 2 (mofettes) are associated with accumulation of carbon from the Earth's mantle. With an integrated approach using isotope geochemistry, soil activity measurements and multi-omics analyses, we demonstrate that high (nearly pure) CO 2 concentrations have strongly affected pathways of carbon production and decomposition and therefore carbon turnover. In particular, a promotion of dark CO 2 fixation significantly increased the input of geogenic carbon in the mofette when compared to a reference wetland soil exposed to normal levels of CO 2 . Radiocarbon analysis revealed that high quantities of mofette soil carbon originated from the assimilation of geogenic CO 2 (up to 67%) via plant primary production and subsurface CO 2 fixation. However, the preservation and accumulation of almost undegraded organic material appeared to be facilitated by the permanent exclusion of meso-to macroscopic eukaryotes and associated physical and/or ecological traits rather than an impaired biochemical potential for soil organic matter decomposition. Our study shows how CO 2 -induced changes in diversity and functions of the soil community can foster an unusual biogeochemical profile.

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Section: Methodsmentioning

confidence: 99%

Altered carbon turnover processes and microbiomes in soils under long-term extremely high CO2 exposure

et al. 2016

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“…A number of limitations need to be considered for the extraction protocol: a) the achievement of total cell lysis, b) co-extraction of inhibitory compounds (e.g., humic acids or polyphenolics), c) contaminating DNA in the RNA fraction, d) rapid degradation of extracted nucleic acids when stored. 23 . To minimize the effect of inhibitory compounds on PCR-based applications 24 , test a range of dilutions from the extracted DNA and RNA.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Tatti

McKew

Whitby

et al. 2016

JoVE

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Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

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“…Shipboard DNA extraction from serpentinite mud samples was unsuccessful using a standard qPCR protocol (e.g., Lever et al, 2015). Additional analysis shows high adsorption of DNA to serpentinite minerals; thus, improvements in DNA extraction protocols in terms of adsorption prevention will be necessary for further DNA extractions.…”

Section: Shipboard Dna Extraction and Qpcrmentioning

confidence: 99%

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Fryer¹,

Wheat²,

Williams³

et al. 2017

International Ocean Discovery Program Preliminary Report

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Geologic processes at convergent plate margins control geochemical cycling, seismicity, and deep biosphere activity in subduction zones and suprasubduction zone lithosphere. International Ocean Discovery Program (IODP) Expedition 366 was designed to address the nature of these processes in the shallow to intermediate depth of the Mariana subduction channel. Although no technology is available to permit direct sampling of the subduction channel of an intraoceanic convergent margin at depths up to 18 km, the Mariana forearc region (between the trench and the active volcanic arc) provides a means to access this zone.Active conduits, resulting from fractures in the forearc, are prompted by along-and across-strike extension that allows slab-derived fluids and materials to ascend to the seafloor along associated faults, resulting in the formation of serpentinite mud volcanoes. Serpentinite mud volcanoes of the Mariana forearc are the largest mud volcanoes on Earth. Their positions adjacent to or atop fault scarps on the forearc are likely related to the regional extension and vertical tectonic deformation in the forearc. Serpentinite mudflows at these volcanoes include serpentinized forearc mantle clasts, crustal and subducted Pacific plate materials, a matrix of serpentinite muds, and deep-sourced formation fluid. Mud volcanism on the Mariana forearc occurs within 100 km of the trench, representing a range of depths and temperatures to the downgoing plate and the subduction channel. These processes have likely been active for tens of millions of years at this site and for billions of years on Earth.At least 10 active serpentinite mud volcanoes have been located in the Mariana forearc. Two of these mud volcanoes are Conical and South Chamorro Seamounts, which are the furthest from the Mariana Trench at 86 and 78 km, respectively. Both seamounts were cored during Ocean Drilling Program (ODP) Legs 125 and 195, respectively. Data from these two seamounts represent deeper, warmer examples of the continuum of slab-derived materials as the Pacific plate subducts, providing a snapshot of how slab subduction affects fluid release, the composition of ascending fluids, mantle hydration, and the metamorphic paragenesis of subducted oceanic lithosphere. Data from the study of these two mud volcanoes constrain the pressure, temperature, and composition of fluids and materials within the subduction channel at depths of about 18 to 19 km. Understanding such processes is necessary for elucidating factors that control seismicity in convergent margins, tectonic and magma genesis processes in the forearc and volcanic arc, fluid and material fluxes, and the nature and variability of environmental conditions that impact subseafloor microbial communities.Expedition 366 centered on data collection from cores recovered from three serpentinite mud volcanoes that define a continuum of subduction-channel processes defined by the two previously cored serpentinite mud volcanoes and the trench. Three serpentinite mud volcanoes (Yinazao, Fa...

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A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

Cited by 271 publications

References 79 publications

Altered carbon turnover processes and microbiomes in soils under long-term extremely high CO2 exposure

Altered carbon turnover processes and microbiomes in soils under long-term extremely high CO2 exposure

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

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