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Improved protocol for the extraction of bacterial mRNA from soils
Journal of Microbiological Methods 91 (2012) 62–64
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Improved protocol for the extraction of bacterial mRNA from soils Shilpi Sharma a, 1, Ravikumar Mehta a, 1, Rashi Gupta a, Michael Schloter b,⁎ a b
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India Research Unit for Environmental Genomics, National Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
a r t i c l e
i n f o
Article history: Received 16 July 2012 Received in revised form 17 July 2012 Accepted 17 July 2012 Available online 27 July 2012
a b s t r a c t An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA. © 2012 Elsevier B.V. All rights reserved.
Keywords: mRNA Soil glnA
High quality RNA is the basis for the analysis of microbial activities in soil. However, the extraction of prokaryotic mRNA from soil is difficult due to various limitations, e.g. the shorter half life time of mRNA compared to rRNA ranging between 30 s and 20 min (Ehretsmann et al., 1992), the contents of mRNA less than 5% in total RNA extracted from soil (Neidhardt and Umbarger, 1996), the absence of poly(A) tail in prokaryotic transcripts as well as fragmented and unstable mRNA because of simultaneous transcription and translation. Furthermore the soil matrix itself can cause additional problems due to the specific sorption of nucleic acids to organic materials and clay particles (Moran et al., 1993). Ribose hydroxyl groups involved in the binding of the ribose can interact with the soil matrix resulting in RNA being more difficult to extract in comparison to DNA (Cleaves et al., 2010). In addition to that, a wide range of inhibitors occurring in soil for enzymatic reactions, especially humic acids, has been known to adversely affect the analysis of mRNA (Moran et al., 1993; Tebbe and Vahjen, 1993). Finally, the ubiquitous occurrence of RNases has been described as a major problem in RNA extraction from soil (Liang and Keeley, 2011). Overall the RNA content of soils ranges between 1.4 and 56 μg RNA g−1 dry weight and is often more related to the soil type, than to general activity pattern of the soil microflora (Hurt et al., 2001). Due to the above reasons there is still a need to improve mRNA extraction protocols from soils for enhanced RNA yields independent from the soil matrix. In this paper we describe an improved extraction protocol for prokaryotic RNA from soils. As a basis the well established protocol by Griffiths et al. (2000) for the co-extraction of DNA and RNA was used. The method involved bead beating of the samples in CTAB extraction buffer followed by phenol-chloroform extraction ⁎ Corresponding author. Tel.: +49 89 3187 2304; fax: +49 89 3187 2800. E-mail address: [email protected] (M. Schloter). 1 Authors contributed equally. 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2012.07.016
and precipitation in PEG. However, the method was developed with emphasis on the co-extraction of DNA and RNA and thus might not be optimally adjusted to the needs of high quality RNA. Moreover, the protocol has been reported to show variability between replicates (Toewe et al., 2011). In the present study, the results of two modifications related to the original protocol are shown, viz. more intensive bead beating for cell lysis, and the addition of PVP for extraction. To prove the suitability of the new approach, the quality of extracted RNA was analyzed spectrometrically and transcripts of the glnA gene, coding for the eubacterial glutamine synthetase, were analyzed. Four different soil samples were collected from agricultural fields in different locations in India in December 2011, and characterized on the basis of texture, pH and humic acid content (Table 1). Three replicates per site (each consisting of four individual soil cores) were sampled from top soil (0–10 cm). Soil samples were shock frozen directly after sampling and stored at −80 °C until nucleic acids were extracted. The extraction of RNA was performed by the method of co-extraction of DNA and RNA as described by Griffiths et al. (2000) with the following modifications: (1) two rounds of bead beating for lysis by adjusting bead beating time and power (in total 5 m s−1 for 40 s), (2) addition of polyvinylpyrrolidone (PVP) to the extraction buffer by mixing equal volumes of 10% (wt/vol) cetyltrimethylammonium bromide (CTAB) and 3.4% PVP in 0.7 M NaCl with 240 mM potassium phosphate buffer (pH 8.0), and (3) nucleic acid precipitation on ice (Zaprasis et al., 2010; Toewe et al., 2011). Nucleic acids were quantified spectrometrically (NanoDrop Technologies, USA) at 260 nm (Sambrook et al., 1989) and the purity was estimated by ratios between the absorbance at 260 nm and 280 nm (A260/A280), and 260 nm and 230 nm (A260/A230). Nucleic acid concentrations in the extracts were calculated assuming an absorbance of 1.0 (10 mm path) at 260 nm, corresponding to a concentration of 46.7 ng μl −1 (Persoh et al., 2008). From the concentration obtained
S. Sharma et al. / Journal of Microbiological Methods 91 (2012) 62–64
63
Table 1 Soil properties. Sample no
Location
Coordinates
Texture
pH
Humic acid (μg g−1 soil)
1
Delhi, India
Sandy loam
8.5
520
2
Hyderabad, India
Sandy
9.7
199
3
Ludhiana, India
Sandy loam
7.2
4
Allahabad, India
29.0167° 77.3833° 17.3667° 78.4667° 30.9100° 75.8500° 25.4500° 81.8500°
Loamy
6.8
N, E N, E N, E N, E
48.56 174.8
nucleic acid yields were calculated per gram dry weight of soil. The concentration of humic acid was measured using spectrometry at 400 nm (Mettel et al., 2010). DNA was removed by treating the samples with DNase I, RNase-free enzyme (Fermentas, USA) as per manufacturer's instructions. RNA was reverse transcribed using the reverse complementary primer GS1β (Hurt et al., 2001) for the eubacterial glnA (glutamine synthetase) gene using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) in a total volume of 20 μl. Aliquots (2 μl) of the reverse transcription products were used for amplification in PCR Master Mix (Bioline, USA) with 10 pmol each of conserved primers GS1β and GS2Υ (Hurt et al., 2001). The amplification conditions included an initial denaturation step of 5 min at 95 °C followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C, and a final extension at 72 °C for 7 min in MJ Mini™ Personal Thermal Cycler (Bio-Rad, USA). The reaction volume was set to 20 μl. RT-PCR amplification products were examined on 1.5% agarose gels. Aliquots (1 μl) of the reverse transcription products were used for real-time PCR in Maxima® SYBR Green/ROX qPCR Master Mix Reagents (Fermentas, USA) with 10 pmol each of conserved primers GS1β and GS2Υ was used for qRT-PCR in a total volume of 10 μl. The reactions were carried out in C1000™ Thermal Cycler (Bio-Rad) in triplicates. The standard curves were prepared with amplicons of glnA generated with DNA from Pseudomonas fluorescens LPK2 as template. Amplification efficiency was found to 86%. No signals were observed in the non-template controls. The data were subjected to analysis of variance (ANOVA) using SPSS Statistical System (SPSS 16.0 for Windows). The comparison between means was made using Duncan's multiple range test (DMRT) at p b 0.05 (Little and Hills, 1978). An increase in the total energy of the initial bead beating resulted in an approx. 56% increase in nucleic acid yields independent from the used soil samples as compared to the original protocol (Griffiths et al., 2000). Bead beating times greater than 5 m s−1 for 40 s however resulted in shearing of nucleic acids as indicated by a reduced quality of 16S rRNA amplicons (data not shown). To reduce the amount of co-extracted humic acids PVP was added to the CTAB based extraction buffer. The addition of PVP to the extraction buffer resulted in a significant decrease (more than half) in humic acid content as compared to the extraction buffer without PVP (Fig. 1a), without compromising with nucleic acid recovery (Fig. 1b). Precipitation of nucleic acids using modified protocol (CTAB/PVP based extraction buffer with additional bead beating) resulted in A260/280 and A260/230 values of 1.96 and 1.89, respectively. While there have been contrasting reports for DNA on the effect of the addition of PVP to the extraction buffer for removal of humic acid on DNA yields (Steffan et al., 1988; Zhou et al., 1996), our results confirm reports by Mettel et al. (2010), who also could verify the high potential of PCP to adsorb humic acids without further losses of RNA. Glutamine synthetase transcripts were employed as marker to confirm the presence of mRNA in the extracts. glnA transcripts were found to be 7 folds higher in the modified protocol as compared to the original protocol by Griffiths et al. (2000), further confirming better recovery of mRNA from the modified protocol (Fig. 2) in all
Fig. 1. Comparison of Griffiths' protocol and the step wise modifications to the same with respect to absorbance at 400 nm for humic acid content (a), nucleic acid yield (μg g−1 dry soil) (b), and A260/280 and A260/230 ratios (c). Error bars represent the standard deviation between the different soil samples analyzed (n = 3 per soil sample). Letters indicate significance levels (p b 0.001).
Fig. 2. Relative number of glnA transcripts using nucleic acid extracted by the original Griffiths' protocol and the modified protocol. Error bars represent the standard deviation between the different soil samples analyzed (n=3 per soil sample). Letters indicate significance levels (pb 0.001).
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S. Sharma et al. / Journal of Microbiological Methods 91 (2012) 62–64
soil samples tested. As expected DNA yields were not affected by the modifications used and glnA gene copy numbers did not vary between the tested extraction protocols. Acknowledgment This research was supported by the Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi. The authors wish to thank Prof T. R. Sreekrishnan for his constant support in carrying out the work. References Cleaves II, H.J., Jonsson, C.M., Sverjensky, D.A., Hazen, R.M., 2010. Adsorption of nucleic acid components on rutile (TiO2) surfaces. Astrobiology 10, 311–323. Ehretsmann, C.P., Carpousis, A.J., Krisch, H.M., 1992. mRNA degradation in prokaryotes. FASEB J. 6, 3186–3192. Griffiths, R.I., Whiteley, A.S., O'Donnell, A.G., Bailey, M.J., 2000. Rapid method for co extraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA‐based microbial community composition. Appl. Environ. Microbiol. 66, 5488–5491. Hurt, A.R., Qiu, X., WU, L., Roh, Y., Palumbo, A.V., Tiedje, J.M., Zhou, J., 2001. Simultaneous recovery of RNA and DNA from soils and sediments. Appl. Environ. Microbiol. 67, 4495–4503. Liang, Z., Keeley, A., 2011. Detection of viable Cryptosporidium parvum in soil by reverse transcription-real-time PCR targeting hsp70 mRNA. Appl. Environ. Microbiol. 77, 6476–6485.
Little, T.M., Hills, F.C., 1978. Agricultural Experimentation. John Wiley and Sons Inc., U.S.A. Mettel, C., Kim, Y., Shrestha, P.M., Liesack, W., 2010. Extraction of mRNA from soil. Appl. Environ. Microbiol. 76, 5995–6000. Moran, M.A., Torsvik, V.L., Torsvik, T., Hodson, R.E., 1993. Direct extraction and purification of rRNA for ecological studies. Appl. Environ. Microbiol. 59, 915–918. Neidhardt, F.C., Umbarger, H.E., 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, pp. 13–16. Persoh, D., Theuerl, S., Buscot, F., Rambold, G., 2008. Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil. J. Microbiol. Meth. 75, 19–24. Sambrook, J., Frotsch, E.F., Maniatis, T., 1989. 2nd ed. Molecular Cloning: A Laboratory Manual, vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Steffan, R.J., Goksoyr, J., Bej, A.K., Atlas, R.M., 1988. Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54, 2908–2915. Tebbe, C.C., Vahjen, W., 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59, 2657–2665. Toewe, S., Wallisch, S., Bannert, A., Fischer, D., Hai, B., Haesler, F., Kleineidam, K., Schloter, M., 2011. Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils. J. Microbiol. Meth. 84, 406–412. Zaprasis, A., Liu, Y.J., Liu, S.J., Drake, H.L., Horn, M.A., 2010. Abundance of novel and diverse tfdA-like genes, encoding putative phenoxyalkanoic acid herbicidedegrading dioxygenases, in soil. Appl. Environ. Microb. 76, 119–128. Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322.
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Improved protocol for the extraction of bacterial mRNA from soils Shilpi Sharma a, 1, Ravikumar Mehta a, 1, Rashi Gupta a, Michael Schloter b,⁎ a b
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India Research Unit for Environmental Genomics, National Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
a r t i c l e
i n f o
Article history: Received 16 July 2012 Received in revised form 17 July 2012 Accepted 17 July 2012 Available online 27 July 2012
a b s t r a c t An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA. © 2012 Elsevier B.V. All rights reserved.
Keywords: mRNA Soil glnA
High quality RNA is the basis for the analysis of microbial activities in soil. However, the extraction of prokaryotic mRNA from soil is difficult due to various limitations, e.g. the shorter half life time of mRNA compared to rRNA ranging between 30 s and 20 min (Ehretsmann et al., 1992), the contents of mRNA less than 5% in total RNA extracted from soil (Neidhardt and Umbarger, 1996), the absence of poly(A) tail in prokaryotic transcripts as well as fragmented and unstable mRNA because of simultaneous transcription and translation. Furthermore the soil matrix itself can cause additional problems due to the specific sorption of nucleic acids to organic materials and clay particles (Moran et al., 1993). Ribose hydroxyl groups involved in the binding of the ribose can interact with the soil matrix resulting in RNA being more difficult to extract in comparison to DNA (Cleaves et al., 2010). In addition to that, a wide range of inhibitors occurring in soil for enzymatic reactions, especially humic acids, has been known to adversely affect the analysis of mRNA (Moran et al., 1993; Tebbe and Vahjen, 1993). Finally, the ubiquitous occurrence of RNases has been described as a major problem in RNA extraction from soil (Liang and Keeley, 2011). Overall the RNA content of soils ranges between 1.4 and 56 μg RNA g−1 dry weight and is often more related to the soil type, than to general activity pattern of the soil microflora (Hurt et al., 2001). Due to the above reasons there is still a need to improve mRNA extraction protocols from soils for enhanced RNA yields independent from the soil matrix. In this paper we describe an improved extraction protocol for prokaryotic RNA from soils. As a basis the well established protocol by Griffiths et al. (2000) for the co-extraction of DNA and RNA was used. The method involved bead beating of the samples in CTAB extraction buffer followed by phenol-chloroform extraction ⁎ Corresponding author. Tel.: +49 89 3187 2304; fax: +49 89 3187 2800. E-mail address: [email protected] (M. Schloter). 1 Authors contributed equally. 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2012.07.016
and precipitation in PEG. However, the method was developed with emphasis on the co-extraction of DNA and RNA and thus might not be optimally adjusted to the needs of high quality RNA. Moreover, the protocol has been reported to show variability between replicates (Toewe et al., 2011). In the present study, the results of two modifications related to the original protocol are shown, viz. more intensive bead beating for cell lysis, and the addition of PVP for extraction. To prove the suitability of the new approach, the quality of extracted RNA was analyzed spectrometrically and transcripts of the glnA gene, coding for the eubacterial glutamine synthetase, were analyzed. Four different soil samples were collected from agricultural fields in different locations in India in December 2011, and characterized on the basis of texture, pH and humic acid content (Table 1). Three replicates per site (each consisting of four individual soil cores) were sampled from top soil (0–10 cm). Soil samples were shock frozen directly after sampling and stored at −80 °C until nucleic acids were extracted. The extraction of RNA was performed by the method of co-extraction of DNA and RNA as described by Griffiths et al. (2000) with the following modifications: (1) two rounds of bead beating for lysis by adjusting bead beating time and power (in total 5 m s−1 for 40 s), (2) addition of polyvinylpyrrolidone (PVP) to the extraction buffer by mixing equal volumes of 10% (wt/vol) cetyltrimethylammonium bromide (CTAB) and 3.4% PVP in 0.7 M NaCl with 240 mM potassium phosphate buffer (pH 8.0), and (3) nucleic acid precipitation on ice (Zaprasis et al., 2010; Toewe et al., 2011). Nucleic acids were quantified spectrometrically (NanoDrop Technologies, USA) at 260 nm (Sambrook et al., 1989) and the purity was estimated by ratios between the absorbance at 260 nm and 280 nm (A260/A280), and 260 nm and 230 nm (A260/A230). Nucleic acid concentrations in the extracts were calculated assuming an absorbance of 1.0 (10 mm path) at 260 nm, corresponding to a concentration of 46.7 ng μl −1 (Persoh et al., 2008). From the concentration obtained
S. Sharma et al. / Journal of Microbiological Methods 91 (2012) 62–64
63
Table 1 Soil properties. Sample no
Location
Coordinates
Texture
pH
Humic acid (μg g−1 soil)
1
Delhi, India
Sandy loam
8.5
520
2
Hyderabad, India
Sandy
9.7
199
3
Ludhiana, India
Sandy loam
7.2
4
Allahabad, India
29.0167° 77.3833° 17.3667° 78.4667° 30.9100° 75.8500° 25.4500° 81.8500°
Loamy
6.8
N, E N, E N, E N, E
48.56 174.8
nucleic acid yields were calculated per gram dry weight of soil. The concentration of humic acid was measured using spectrometry at 400 nm (Mettel et al., 2010). DNA was removed by treating the samples with DNase I, RNase-free enzyme (Fermentas, USA) as per manufacturer's instructions. RNA was reverse transcribed using the reverse complementary primer GS1β (Hurt et al., 2001) for the eubacterial glnA (glutamine synthetase) gene using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) in a total volume of 20 μl. Aliquots (2 μl) of the reverse transcription products were used for amplification in PCR Master Mix (Bioline, USA) with 10 pmol each of conserved primers GS1β and GS2Υ (Hurt et al., 2001). The amplification conditions included an initial denaturation step of 5 min at 95 °C followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C, and a final extension at 72 °C for 7 min in MJ Mini™ Personal Thermal Cycler (Bio-Rad, USA). The reaction volume was set to 20 μl. RT-PCR amplification products were examined on 1.5% agarose gels. Aliquots (1 μl) of the reverse transcription products were used for real-time PCR in Maxima® SYBR Green/ROX qPCR Master Mix Reagents (Fermentas, USA) with 10 pmol each of conserved primers GS1β and GS2Υ was used for qRT-PCR in a total volume of 10 μl. The reactions were carried out in C1000™ Thermal Cycler (Bio-Rad) in triplicates. The standard curves were prepared with amplicons of glnA generated with DNA from Pseudomonas fluorescens LPK2 as template. Amplification efficiency was found to 86%. No signals were observed in the non-template controls. The data were subjected to analysis of variance (ANOVA) using SPSS Statistical System (SPSS 16.0 for Windows). The comparison between means was made using Duncan's multiple range test (DMRT) at p b 0.05 (Little and Hills, 1978). An increase in the total energy of the initial bead beating resulted in an approx. 56% increase in nucleic acid yields independent from the used soil samples as compared to the original protocol (Griffiths et al., 2000). Bead beating times greater than 5 m s−1 for 40 s however resulted in shearing of nucleic acids as indicated by a reduced quality of 16S rRNA amplicons (data not shown). To reduce the amount of co-extracted humic acids PVP was added to the CTAB based extraction buffer. The addition of PVP to the extraction buffer resulted in a significant decrease (more than half) in humic acid content as compared to the extraction buffer without PVP (Fig. 1a), without compromising with nucleic acid recovery (Fig. 1b). Precipitation of nucleic acids using modified protocol (CTAB/PVP based extraction buffer with additional bead beating) resulted in A260/280 and A260/230 values of 1.96 and 1.89, respectively. While there have been contrasting reports for DNA on the effect of the addition of PVP to the extraction buffer for removal of humic acid on DNA yields (Steffan et al., 1988; Zhou et al., 1996), our results confirm reports by Mettel et al. (2010), who also could verify the high potential of PCP to adsorb humic acids without further losses of RNA. Glutamine synthetase transcripts were employed as marker to confirm the presence of mRNA in the extracts. glnA transcripts were found to be 7 folds higher in the modified protocol as compared to the original protocol by Griffiths et al. (2000), further confirming better recovery of mRNA from the modified protocol (Fig. 2) in all
Fig. 1. Comparison of Griffiths' protocol and the step wise modifications to the same with respect to absorbance at 400 nm for humic acid content (a), nucleic acid yield (μg g−1 dry soil) (b), and A260/280 and A260/230 ratios (c). Error bars represent the standard deviation between the different soil samples analyzed (n = 3 per soil sample). Letters indicate significance levels (p b 0.001).
Fig. 2. Relative number of glnA transcripts using nucleic acid extracted by the original Griffiths' protocol and the modified protocol. Error bars represent the standard deviation between the different soil samples analyzed (n=3 per soil sample). Letters indicate significance levels (pb 0.001).
64
S. Sharma et al. / Journal of Microbiological Methods 91 (2012) 62–64
soil samples tested. As expected DNA yields were not affected by the modifications used and glnA gene copy numbers did not vary between the tested extraction protocols. Acknowledgment This research was supported by the Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi. The authors wish to thank Prof T. R. Sreekrishnan for his constant support in carrying out the work. References Cleaves II, H.J., Jonsson, C.M., Sverjensky, D.A., Hazen, R.M., 2010. Adsorption of nucleic acid components on rutile (TiO2) surfaces. Astrobiology 10, 311–323. Ehretsmann, C.P., Carpousis, A.J., Krisch, H.M., 1992. mRNA degradation in prokaryotes. FASEB J. 6, 3186–3192. Griffiths, R.I., Whiteley, A.S., O'Donnell, A.G., Bailey, M.J., 2000. Rapid method for co extraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA‐based microbial community composition. Appl. Environ. Microbiol. 66, 5488–5491. Hurt, A.R., Qiu, X., WU, L., Roh, Y., Palumbo, A.V., Tiedje, J.M., Zhou, J., 2001. Simultaneous recovery of RNA and DNA from soils and sediments. Appl. Environ. Microbiol. 67, 4495–4503. Liang, Z., Keeley, A., 2011. Detection of viable Cryptosporidium parvum in soil by reverse transcription-real-time PCR targeting hsp70 mRNA. Appl. Environ. Microbiol. 77, 6476–6485.
Little, T.M., Hills, F.C., 1978. Agricultural Experimentation. John Wiley and Sons Inc., U.S.A. Mettel, C., Kim, Y., Shrestha, P.M., Liesack, W., 2010. Extraction of mRNA from soil. Appl. Environ. Microbiol. 76, 5995–6000. Moran, M.A., Torsvik, V.L., Torsvik, T., Hodson, R.E., 1993. Direct extraction and purification of rRNA for ecological studies. Appl. Environ. Microbiol. 59, 915–918. Neidhardt, F.C., Umbarger, H.E., 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, pp. 13–16. Persoh, D., Theuerl, S., Buscot, F., Rambold, G., 2008. Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil. J. Microbiol. Meth. 75, 19–24. Sambrook, J., Frotsch, E.F., Maniatis, T., 1989. 2nd ed. Molecular Cloning: A Laboratory Manual, vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Steffan, R.J., Goksoyr, J., Bej, A.K., Atlas, R.M., 1988. Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54, 2908–2915. Tebbe, C.C., Vahjen, W., 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59, 2657–2665. Toewe, S., Wallisch, S., Bannert, A., Fischer, D., Hai, B., Haesler, F., Kleineidam, K., Schloter, M., 2011. Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils. J. Microbiol. Meth. 84, 406–412. Zaprasis, A., Liu, Y.J., Liu, S.J., Drake, H.L., Horn, M.A., 2010. Abundance of novel and diverse tfdA-like genes, encoding putative phenoxyalkanoic acid herbicidedegrading dioxygenases, in soil. Appl. Environ. Microb. 76, 119–128. Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316–322.