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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.


Electrophoretic profiles (agarose 1%) of 10 μL of the final RNA extract (step 15b of the protocol), and 12 μL of the 1 kb ladder (Invitrogen, France). Gel was stained with ethidium bromide.
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fig0030: Electrophoretic profiles (agarose 1%) of 10 μL of the final RNA extract (step 15b of the protocol), and 12 μL of the 1 kb ladder (Invitrogen, France). Gel was stained with ethidium bromide.

Mentions: The DNA and RNA were quantified by fluorometry using the Quant-iT™ Pico Green DNA and Quant-iT™ Ribo Green RNA assay kit (Molecular Probes, Carlsbad, New Mexico) respectively in accordance with the manufacturer’s instructions. The purity and integrity of the RNA recovered was also verified after migration on a 1% agarose gel (Fig. 6).


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)

Electrophoretic profiles (agarose 1%) of 10 μL of the final RNA extract (step 15b of the protocol), and 12 μL of the 1 kb ladder (Invitrogen, France). Gel was stained with ethidium bromide.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0030: Electrophoretic profiles (agarose 1%) of 10 μL of the final RNA extract (step 15b of the protocol), and 12 μL of the 1 kb ladder (Invitrogen, France). Gel was stained with ethidium bromide.
Mentions: The DNA and RNA were quantified by fluorometry using the Quant-iT™ Pico Green DNA and Quant-iT™ Ribo Green RNA assay kit (Molecular Probes, Carlsbad, New Mexico) respectively in accordance with the manufacturer’s instructions. The purity and integrity of the RNA recovered was also verified after migration on a 1% agarose gel (Fig. 6).

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.