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Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA simultaneously from soil, without the use of harmful solvents.

Tournier E, Amenc L, Pablo AL, Legname E, Blanchart E, Plassard C, Robin A, Bernard L - MethodsX (2015)

Bottom Line: An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents.The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. •A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters.•The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult.•The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content.

View Article: PubMed Central - PubMed

Affiliation: IRD, UMR Eco&Sols, 2 Place Viala, 34060 Montpellier Cedex 1, France.

ABSTRACT
An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents. The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. •A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters.•The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult.•The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content.

No MeSH data available.


(a) Bacterial (16S) and (b) fungal (18S) ribosomal gene copy numbers per gram of soil, measured by qPCR as a function of the soil homogenization treatment before subsampling (0.25 g and 0.50 g of soil) for the co-extraction protocol. 30 g of soil were used per homogenization treatment. Errors bars correspond to 95% confidence intervals (alpha 0.05, 3 replicates).
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fig0035: (a) Bacterial (16S) and (b) fungal (18S) ribosomal gene copy numbers per gram of soil, measured by qPCR as a function of the soil homogenization treatment before subsampling (0.25 g and 0.50 g of soil) for the co-extraction protocol. 30 g of soil were used per homogenization treatment. Errors bars correspond to 95% confidence intervals (alpha 0.05, 3 replicates).

Mentions: For the analyses of fungal parameters (density and diversity), a sample size of 1 g has been recommended in order to be representative of the community [4]. Therefore, the applicability to fungal biomass quantification (see method below) was tested by crushing and homogenizing 30 g of soil in liquid nitrogen either by hand, or using a mortar grinder (Pulverisette 2, Fritsch, Idar-Oberstein, Germany) prior to subsample 0.25 g and then beginning the protocol. From our results, it appears that crushing 30 g of soil in liquid nitrogen prior to subsample 0.25 g for the further co-extraction significantly improved the recovery of 18S genes copies from the DNA while it has no effect on the 16S copy number (Fig. 7). The quantitative PCR protocol used is described in Supplementary material (S1).


Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA simultaneously from soil, without the use of harmful solvents.

Tournier E, Amenc L, Pablo AL, Legname E, Blanchart E, Plassard C, Robin A, Bernard L - MethodsX (2015)

(a) Bacterial (16S) and (b) fungal (18S) ribosomal gene copy numbers per gram of soil, measured by qPCR as a function of the soil homogenization treatment before subsampling (0.25 g and 0.50 g of soil) for the co-extraction protocol. 30 g of soil were used per homogenization treatment. Errors bars correspond to 95% confidence intervals (alpha 0.05, 3 replicates).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487712&req=5

fig0035: (a) Bacterial (16S) and (b) fungal (18S) ribosomal gene copy numbers per gram of soil, measured by qPCR as a function of the soil homogenization treatment before subsampling (0.25 g and 0.50 g of soil) for the co-extraction protocol. 30 g of soil were used per homogenization treatment. Errors bars correspond to 95% confidence intervals (alpha 0.05, 3 replicates).
Mentions: For the analyses of fungal parameters (density and diversity), a sample size of 1 g has been recommended in order to be representative of the community [4]. Therefore, the applicability to fungal biomass quantification (see method below) was tested by crushing and homogenizing 30 g of soil in liquid nitrogen either by hand, or using a mortar grinder (Pulverisette 2, Fritsch, Idar-Oberstein, Germany) prior to subsample 0.25 g and then beginning the protocol. From our results, it appears that crushing 30 g of soil in liquid nitrogen prior to subsample 0.25 g for the further co-extraction significantly improved the recovery of 18S genes copies from the DNA while it has no effect on the 16S copy number (Fig. 7). The quantitative PCR protocol used is described in Supplementary material (S1).

Bottom Line: An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents.The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. •A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters.•The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult.•The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content.

View Article: PubMed Central - PubMed

Affiliation: IRD, UMR Eco&Sols, 2 Place Viala, 34060 Montpellier Cedex 1, France.

ABSTRACT
An optimized method, based on the coupling of two commercial kits, is described for the extraction of soil nucleic acids, with simultaneous extraction and purification of DNA and RNA following a cascade scheme and avoiding the use of harmful solvents. The protocol canmonitor the variations in the recovery yield of DNA and RNA from soils of various types.The quantitative version of the protocol was obtained by testing the starting soil quantity, the grinding parameters and the final elution volumes, in order to avoid saturation of both kits. •A first soil-crushing step in liquid nitrogen could be added for the assessment of fungal parameters.•The protocol was efficienton different tropical soils, including Andosol, while their high contents of clays, including poorly crystalline clays, and Fe and Al oxides usually make the nucleic acid extraction more difficult.•The RNA recovery yield from the previous tropical soils appeared to correlate better to soil respiration than DNA, which is positively influenced by soil clay content.

No MeSH data available.