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A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria
FEMS Microbiology Letters 229 (2003) 97^101
www.fems-microbiology.org
A simple and e⁄cient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria Kidon Sung, Saeed A. Khan , Mohamed S. Nawaz, Ashraf A. Khan U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, 3900 NCTR Road, Je¡erson, AR 72079, USA Received 21 August 2003; accepted 15 October 2003 First published online 6 November 2003
Abstract A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2^2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gramnegative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments. 8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : RNA; Electrophoresis; Hybridization
1. Introduction A number of methods have been reported for the isolation of RNA from Gram-positive and Gram-negative bacteria [1^5]. These methods involve enzymatic lysis [1], sonication [2], bead beating [3], cesium chloride precipitation [4], and treatment with guanidine isothiocyanate, phenol and sodium dodecyl sulfate (SDS) to inhibit RNases [5]. These methods are all time-consuming, laborious and costly. Moreover, the RNA preparations are loaded with s 70% rRNA and 10^15% tRNA [6]. Only a minor population of the RNA obtained (3^5%) represents the mRNA. The rRNA and tRNA are of no use in various molecular biology applications such as cloning, reverse transcription (RT)/RT-polymerase chain reaction (PCR),
* Corresponding author. Tel. : +1 (870) 543-7197; Fax : +1 (870) 543-7307. E-mail address : [email protected] (S.A. Khan).
and gene-expression assays. To free bacterial RNA preparations from contaminating rRNA, modi¢ed versions of nucleic acid capture techniques [7^9] are used. These involve binding of rRNA to a capture oligonucleotide that contains sequences complementary to 16S and 23S rRNA at one end and sequences at the other end complementary to the sequences attached to magnetic beads. After the binding of 16S and 23S rRNA to capture oligonucleotide, the capture oligo-16S/23S complex is then incubated with oligonucleotide-coated magnetic beads that bind to the complementary sequences at the other end of the capture oligonucleotides. This complex is then magnetically removed leaving the sample free of 16S and 23S rRNA. This, however, adds to the cost of obtaining high-quality RNA. The method reported here yields a good-quality RNA that is free from 16S and 23S rRNA, contains full-length transcripts and does not require costly and additional puri¢cation steps. The method is applicable for the isolation of RNA from at least eight bacterial genera belonging to Gram-positive and Gram-negative categories.
0378-1097 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00791-2
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2. Materials and methods 2.1. Bacterial strains and culture conditions Five Gram-positive (Enterococcus faecium ATCC 51559, Staphylococcus aureus, Lactococcus lactis, Lactobacillus reuteri, Mycobacterium vanbaalenii DSM 7251) and four Gram-negative bacteria (Escherichia coli XLOLR, Salmonella typhimurium DT8, Aeromonas veronii, and Campylobacter jejuni) were used for RNA isolation. Bacteria were maintained as in-house stocks. E. faecium was obtained from ATCC and S. typhimurium was obtained from Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, Washington, DC, USA. Most of the bacteria were grown aerobically for 4^8 h at 37‡C in brain heart infusion (BHI) medium (BHI, 6 g; peptic digest of animal tissue, 7 g; pancreatic digest of casein, 14.5 g; NaCl, 5 g; dextrose, 2 g; and disodium phosphate, 2.5 g, l31 ). However, M. vanbaalenii was grown at 30‡C for 48 h because of its slower growth. Campylobacter was grown on Campy agar plates (Remel) at 37‡C for 48^72 h under microaerophilic conditions (89% N2 , 5% CO2 , and 6% O2 ) [14]. Lactococci and lactobacilli were grown in MRS broth [15] for 18 h at 37‡C. 2.2. RNA isolation by the Qiagen method Exponentially growing bacteria from liquid media or from agar plates were suspended in TE (10 mM Tris^ HCl, 1 mM ethylenediamine tetraacetic acid (EDTA), pH 7.5) bu¡er and adjusted to a cell density of 5U108 cells per ml. A 10-ml cell suspension was used for all RNA isolation procedures. Centrifugation was carried out at 5000Ug for 5 min at 4‡C unless noted otherwise. RNA isolation and DNase treatment by the Qiagen method were carried out as per the manufacturer’s instructions. The quantity of RNA was determined by measuring the absorbance at 260 nm, A260 (optical density (OD) 1 at A260 = 40 Wg ml31 ), using a Smartspec 3000 spectrophotometer (Bio-Rad), and its purity was determined by measuring the A260 /A280 ratio. 2.3. RNA isolation by the hot phenol extraction method A 10-ml culture was added to a tube containing 1.25 ml of ice-cold ethanol/phenol stop solution (5% water-saturated phenol, pH 6 7.0, in 95% ethanol). RNA isolation and DNase treatment were carried out as described (http://www.microarrays.org/pdfs/Total_RNA_from_Ecoli.pdf). The RNA was suspended in 50 Wl of RNase-free water and the A260 and A280 values were determined. 2.4. RNA extraction by the Triton X-100 boiling method After centrifugation, the pellet was washed with 5 ml of TE. The cells were suspended in 1 ml of TE bu¡er con-
taining 0.2% Triton X-100 (Sigma). The sample was boiled at 100‡C for 10 min and then transferred to an ice bath. An equal volume of chloroform alone or a chloroform^ methanol (1:2 or 2:1) mixture was added. The tubes were inverted 10^15 times and then centrifuged at 12 000Ug at 4‡C for 10 min. The aqueous phase was collected and treated once more with an equal volume of chloroform. After centrifugation, the RNA was then precipitated by the addition of 1/10 volume of 3 M sodium acetate, pH 5.2, 2^2.5 volumes of prechilled absolute ethanol, and incubation at 320‡C for 20 min to overnight. The precipitate was collected by centrifugation at 12 000Ug for 10 min at 4‡C. The pellet was washed twice with 1 ml of 70% ethanol and once with absolute ethanol. After centrifugation, the pellet was air-dried for 5 min and dissolved in 200 Wl of diethylpyrocarbonate (DEPC)-treated water. DNase treatment was done as described above. 2.5. RT-PCR analysis To check the suitability of RNA for RT-PCR analysis, we isolated RNA by the Triton X-100 boiling method from vancomycin-resistant E. faecium ATCC strain 51559. It was divided in three aliquots. One of the RNA aliquots was treated with DNase, the other with DNase and RNase and the third was left untreated. These RNA samples were then used to check for vanA gene-speci¢c mRNA by using vanA gene-speci¢c forward (5P-GGGAAAACGACAATTGC-3P) and reverse (5P-GTACAATGCGGCCGTTA-3P) PCR primers [11] and a one-step RT-PCR kit (Invitrogen). The reaction was carried out in 50 Wl of 1Uproprietary reaction mix containing 0.2 mM each of deoxyribonucleoside triphosphate (dNTP), 1.2 mM of MgSO4 , 0.4 WM each of the forward and reverse primers, 10 Wg of RNA, and 2.5 units of RT/platinum Taq mix. The cycling conditions were as follows: One cycle at 48‡C for 30 min (to copy cDNA from vanA genespeci¢c mRNA) followed by 94‡C denaturation for 2 min; 20 cycles at 94‡C, 15 s, 65^55‡C, 30 s (30.5‡C decrement for each cycle), 68‡C, 30 s. 15 more cycles were carried out at 94‡C, 15 s, 53‡C, 30 s, 68‡C, 30 s; and the last cycle at 68‡C, 420 s. After the cycling, samples were stored at 4‡C until electrophoresis. 2.6. Agarose gel electrophoresis The integrity of an RNA sample was tested by separating 0.2^1 Wg of RNA on a 1.2% agarose gel containing 0.66 M formaldehyde. The gels were run in MOPS electrophoresis bu¡er (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) for 3 h at 90 mV [10]. The gels were stained in ethidium bromide (1 Wg ml31 in electrophoresis bu¡er) and photographed using a gel documentation system, GDS 8000 (UVP, Inc.). PCR-ampli¢ed DNA fragment(s) were separated on a 1.5% agarose gel [12].
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Fig. 1. Comparison of RNA isolation methods. 1 Wg of RNA from E. faecium ATCC 51559 was loaded on a 1.2% agarose gel and run as described in Section 2. Lanes 1 and 7, RNA ladder; lane 2, total RNA by the Qiagen method ; lane 3, RNA by the boiling method (chloroform extraction) ; lane 4, RNA by the boiling method (chloroform^methanol (1:2) extraction); lane 5, RNA by the boiling method (chloroform^ methanol (2:1) extraction); lane 6, RNA by the hot phenol extraction method.
2.7. Northern blotting, probe labeling and hybridization For the detection of full-length transcripts of the vanA gene, 2 Wg of RNA was separated on an agarose gel and transferred to a Hybond-N membrane by vacuum blotting. The nylon membrane was subjected to hybridization with a digoxigenin (DIG)-labeled partial PCR product of the vanA gene. Probe labeling, hybridization, washing and signal detection were carried out as described [13].
3. Results and discussion Most methods for RNA isolation are time-consuming, costly, and cumbersome [1^5]. Moreover, the RNA obtained by these methods is also contaminated with s 70% of 16S and 23S rRNAs [6]. Removal of contami-
99
nating rRNA often requires an extra puri¢cation step using nucleic acid capture techniques [7^9]. The Qiagen and hot phenol extraction methods yielded RNA that was contaminated with 16S and 23S rRNA (Fig. 1, lanes 2 and 6) that needed to be removed before it could be used for molecular biology applications. However, the Triton X100 boiling method reported here provided good-quality RNA (free from contaminating 16S and 23S rRNA) (Fig. 1, lanes 3^5), contained full-length transcripts (Fig. 4) and did not require additional puri¢cation. The absence of rRNA in this preparation is probably due to heat degradation as observed earlier [16]. The quantity of RNA obtained by the boiling method ranged from 0.2 to 2 mg ml31 and had an A260 /A280 ratio between 1.9 and 2.0, which indicated a good quality of RNA (Table 1). A comparison of the A260 /A280 ratios and the yield of RNA obtained by di¡erent methods suggested comparable quality and yield (Table 1). Treatment of the boiled cells with chloroform or a chloroform^methanol mixture had no signi¢cant e¡ect on the yield or quality of RNA (Fig. 1, lanes 3^5) in triplicate experiments. RNA obtained by the Qiagen and hot phenol extraction methods was, however, contaminated with 16S and 23S rRNA (Fig. 1, lanes 2 and 6). Assuming at least 70% contamination with rRNA [6] and that 100% recovery is made in subsequent puri¢cation steps by the oligonucleotide capture method, the e¡ective yield of RNA by these methods would be only one-third of the Triton X-100 boiling method reported here. Therefore, our method is quantitatively superior to the Qiagen and hot phenol extraction methods. The e⁄cacy of this method to isolate RNA from di¡erent bacterial genera and species was demonstrated by successful isolation of RNA from ¢ve Gram-positive bacteria and four Gram-negative bacteria (Fig. 2). The quantity of RNA varied (0.2^2 mg ml31 ) depending upon the bacterial genera and species (Fig. 2, lanes 3, 8^10, and Table 1) but no di¡erences were obtained in the quality of RNA as shown by the gel patterns (Fig. 2) and A260 /A280 ratio (Table 1). A smear between 23S and 5S RNA was observed in all the RNA samples irrespective of the method
Table 1 Comparison of A260 /A280 ratios and the yield of RNA by di¡erent methods Bacteria
Gram-positive E. faecium S. aureus L. lactis L. reuteri M. vanbaalenii Gram-negative E. coli XLOLR S. typhimurium A. veronii C. jejuni
Qiagen method
Hot phenol method
A260 /A280
Yield (mg ml
2.03 2.05 1.99 1.97 1.96 2.03 1.92 2.03 2.01
)
Triton X-100 boiling method
A260 /A280
Yield (mg ml
1.02 0.92 0.65 0.83 0.22
1.99 1.98 2.01 2.03 1.96
2.40 0.20 2.40 0.23
1.99 1.96 2.01 2.01
31
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A260 /A280
Yield (mg ml31 )
0.95 1.02 0.69 0.92 0.22
2.01 2.03 2.05 1.98 1.98
1.01 0.95 0.72 0.88 0.25
2.32 0.22 2.12 0.22
1.99 1.97 2.02 1.99
2.10 0.22 1.99 0.26
31
)
100
K. Sung et al. / FEMS Microbiology Letters 229 (2003) 97^101
used for RNA isolation (Fig. 1, lanes 2^6). It is di⁄cult to tell whether it represents the degraded RNA or di¡erent size mRNA species or both. RNA preparations from E. faecium and L. reuteri also show some bands between 23S and 5S RNA (Fig. 2, lanes 2 and 5). These bands were not observed in other bacterial species (Fig. 2). At least three repetitions of the same experiment (Fig. 2) gave similar results. Since all the RNA preparations were treated with DNase, the possibility that the DNA might be contributing to the bands observed in RNA samples from E. faecium and L. reuteri was ruled out. Moreover, the presence of a 732-bp partially ampli¢ed RT-PCR product of the vanA gene from DNase-treated RNA sample of vancomycin-resistant E. faecium ATCC 51559 (Fig. 3, lane 3) also demonstrated the absence of DNA contami-
nation. However, it can also be contemplated that an incomplete removal of DNA from DNase-digested RNA could be the source of ampli¢cation. This was further disregarded by the observation that no ampli¢cation product was observed after gel analysis when DNase-treated RNA sample was further treated with RNase and used as a template in an RT-PCR reaction (Fig. 3, lane 4). Partial ampli¢cation of the vanA gene from RNA (Fig. 3, lanes 2 and 3) clearly indicated vanA gene-speci¢c mRNA (Fig. 3, lane 3). Ampli¢cation was also observed when the RNA isolated from E. faecium by hot phenol and Qiagen RNA isolation methods was used in an RT-PCR reaction (data not shown). Although, partial ampli¢cation of the vanA gene was observed from RNA irrespective of the isolation method, it did not demonstrate the integrity of the full-length transcript. To detect a full-length transcript of vanA gene-speci¢c mRNA (1031 bp), Northern-blotted RNA from E. faecium was probed with a DIG-labeled vanA PCR probe. A speci¢c hybridization signal in the region where a full-length transcript of the vanA gene would be present (Fig. 4B, lane 2) was observed, suggesting that the integrity of the RNA sample was maintained by use of this method. Boiling has been shown to degrade the rRNA [16], but no information was provided about the fate of mRNA. This study clearly demonstrates that the mRNA is protected. This is also supported by a previous study [17] where environmental samples of bacteria were collected on ¢lters and RNA was isolated by boiling in the presence of SDS^DEPC followed by acid-guanidinium extraction. The integrity of RNA was demonstrated by the ampli¢cation of various full-length naphthalene-degrading genes using RT-PCR. The Triton X-100 boiling method does not involve elaborate RNA isolation procedures, is rapid, and quantitatively yields more RNA than other methods reported here. The simplicity of the procedure, its usefulness for the isolation of RNA from Gram-
Fig. 3. Ampli¢cation of the partial vanA gene by RT-PCR. Lanes 1 and 5, 100-bp DNA ladder; lane 2, RT-PCR from DNase-untreated sample; lane 3, RT-PCR from DNase-treated RNA sample; and lane 4, RTPCR from DNase- and RNase-treated RNA sample.
Fig. 4. Northern blot analysis of full-length vanA gene transcript. Lanes 1 and 3, RNA ladder ; lane 2, RNA from E. faecium ATCC 51559 (2 Wg). A: Ethidium bromide-stained gel. B: Northern blot of gel in A probed with a DIG-labeled partial vanA gene PCR product.
Fig. 2. RNA isolation from di¡erent bacteria by the boiling chloroform extraction method. The amount of RNA loaded in each lane is indicated in parentheses after the bacterial species. Lanes 1 and 11, RNA ladder; lane 2, E. faecium (0.5 Wg); lane 3, S. aureus (0.5 Wg); lane 4, L. reuteri (1 Wg); lane 5, L. lactis (1 Wg); lane 6, A. veronii (1 Wg); lane 7, E. coli (1 Wg); lane 8, C. jejuni (0.2 Wg); lane 9, S. typhimurium (0.2 Wg); and lane 10, M. vanbaalenii (0.2 Wg).
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positive and Gram-negative bacteria, the absence of contaminating 16S and 23S rRNA, and the presence of fulllength transcripts make the Triton X-100 boiling method more desirable than other methods.
[7]
[8]
Acknowledgements The work was supported in part by the postgraduate research program at the National Center for Toxicological Research (NCTR) administered by the Oak Ridge Institute for Science and Education through an inter-agency agreement between the U.S. Department of Energy and U.S. Food and Drug Administration. We thank Drs. John Sutherland and Doug Wagner at NCTR for their critical evaluation of the manuscript.
[9]
[10]
[11]
[12]
References [1] Bashyam, M.D. and Tyagi, A. (1994) An e⁄cient and high-yielding method for isolation of RNA from mycobacteria. BioTechniques 17, 834^836. [2] Rajagopalan, M., Boggaram, V. and Madiraju, M.V. (1995) A rapid protocol for the isolation of RNA from mycobacteria. Lett. Appl. Microbiol. 21, 14^17. [3] Santos, A.R., De Miranda, A.B., Sarno, E.N., Su¡ys, P.N. and Degrave, W.M. (1993) Use of PCR-mediated ampli¢cation of Mycobacterium leprae DNA in di¡erent types of clinical samples for the diagnosis of leprosy. J. Med. Microbiol. 39, 298^304. [4] Glisin, V., Crkvenjakov, R. and Byus, C. (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 2633^ 2637. [5] Bernstein, J.A., Khodursky, A.B., Lin, P.H., Lin-Chao, S. and Cohen, S. (2002) Global analysis of mRNA decay and abundance in E. coli at a single-gene resolution using two-color £uorescent DNA microarrays. Proc. Natl. Acad. Sci. USA 99, 9697^9702. [6] Neidhardt, F.C. and Umbarger, H.E. (1996) In: Escherichia coli and
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[14]
[15] [16]
[17]
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Salmonella (Neidhardt, F.C., Ed.), Vol. 1, pp. 13^16. ASM Press, Washington, DC. Morrissey, D.V. and Collins, M.L. (1989) Nucleic acid hybridization assays employing dA-tailed capture probes: Single capture methods. Mol. Cell. Probes 3, 189^207. Morrissey, D.V., Lombardo, M., Eldredge, J.K., Kearney, K.R., Groody, E.P. and Collins, M.L. (1989) Nucleic acid hybridization assays employing dA-tailed capture probes. I. Multiple capture methods. Anal. Biochem. 18, 345^359. Hunsaker, W.R., Badri, H., Lombardo, M. and Collins, M.L. (1989) Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods. Anal. Biochem. 18, 360^370. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Dutka-Malen, S., Evers, S. and Courvalin, P. (1995) Detection of glycopeptide resistance genotypes and identi¢cation to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33, 24^27. Rajesh, N., Khan, S.A., Watson, R.H. and Cerniglia, C.E. (2002) In£uence of growth media on vancomycin resistance of Enterococcus isolates and correlation with resistance gene determinants. FEMS Microbiol. Lett. 214, 159^163. Khan, S.A., Nawaz, M.S., Khan, A.A. and Cerniglia, C.E. (1999) Direct in-gel hybridization of digoxigenin-labeled non-radioactive probes. Mol. Cell. Probes 13, 233^237. Giesendorf, B.A., Quint, W.G.V., Henkins, M.H.C., Stegeman, H., Huf, F.A. and Neisters, H.G.M. (1992) Rapid and sensitive detection of Campylobacter spp. in chicken products by using polymerase chain reaction. Appl. Environ. Microbiol. 58, 3804^3808. De Man, J.C., Rogosa, M. and Sharpe, M.E. (1960) A medium for cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130. McKillip, J.L., Jaykus, L.A. and Drake, M.A. (1998) rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7. Appl. Environ. Microbiol. 64, 4264^4268. Wilson, M.S., Bakermans, C. and Madsen, E.L. (1999) In situ, realtime catabolic gene expression : extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater. Appl. Environ. Microbiol. 65, 80^87.
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A simple and e⁄cient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria Kidon Sung, Saeed A. Khan , Mohamed S. Nawaz, Ashraf A. Khan U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, 3900 NCTR Road, Je¡erson, AR 72079, USA Received 21 August 2003; accepted 15 October 2003 First published online 6 November 2003
Abstract A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2^2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gramnegative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments. 8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : RNA; Electrophoresis; Hybridization
1. Introduction A number of methods have been reported for the isolation of RNA from Gram-positive and Gram-negative bacteria [1^5]. These methods involve enzymatic lysis [1], sonication [2], bead beating [3], cesium chloride precipitation [4], and treatment with guanidine isothiocyanate, phenol and sodium dodecyl sulfate (SDS) to inhibit RNases [5]. These methods are all time-consuming, laborious and costly. Moreover, the RNA preparations are loaded with s 70% rRNA and 10^15% tRNA [6]. Only a minor population of the RNA obtained (3^5%) represents the mRNA. The rRNA and tRNA are of no use in various molecular biology applications such as cloning, reverse transcription (RT)/RT-polymerase chain reaction (PCR),
* Corresponding author. Tel. : +1 (870) 543-7197; Fax : +1 (870) 543-7307. E-mail address : [email protected] (S.A. Khan).
and gene-expression assays. To free bacterial RNA preparations from contaminating rRNA, modi¢ed versions of nucleic acid capture techniques [7^9] are used. These involve binding of rRNA to a capture oligonucleotide that contains sequences complementary to 16S and 23S rRNA at one end and sequences at the other end complementary to the sequences attached to magnetic beads. After the binding of 16S and 23S rRNA to capture oligonucleotide, the capture oligo-16S/23S complex is then incubated with oligonucleotide-coated magnetic beads that bind to the complementary sequences at the other end of the capture oligonucleotides. This complex is then magnetically removed leaving the sample free of 16S and 23S rRNA. This, however, adds to the cost of obtaining high-quality RNA. The method reported here yields a good-quality RNA that is free from 16S and 23S rRNA, contains full-length transcripts and does not require costly and additional puri¢cation steps. The method is applicable for the isolation of RNA from at least eight bacterial genera belonging to Gram-positive and Gram-negative categories.
0378-1097 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00791-2
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2. Materials and methods 2.1. Bacterial strains and culture conditions Five Gram-positive (Enterococcus faecium ATCC 51559, Staphylococcus aureus, Lactococcus lactis, Lactobacillus reuteri, Mycobacterium vanbaalenii DSM 7251) and four Gram-negative bacteria (Escherichia coli XLOLR, Salmonella typhimurium DT8, Aeromonas veronii, and Campylobacter jejuni) were used for RNA isolation. Bacteria were maintained as in-house stocks. E. faecium was obtained from ATCC and S. typhimurium was obtained from Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, Washington, DC, USA. Most of the bacteria were grown aerobically for 4^8 h at 37‡C in brain heart infusion (BHI) medium (BHI, 6 g; peptic digest of animal tissue, 7 g; pancreatic digest of casein, 14.5 g; NaCl, 5 g; dextrose, 2 g; and disodium phosphate, 2.5 g, l31 ). However, M. vanbaalenii was grown at 30‡C for 48 h because of its slower growth. Campylobacter was grown on Campy agar plates (Remel) at 37‡C for 48^72 h under microaerophilic conditions (89% N2 , 5% CO2 , and 6% O2 ) [14]. Lactococci and lactobacilli were grown in MRS broth [15] for 18 h at 37‡C. 2.2. RNA isolation by the Qiagen method Exponentially growing bacteria from liquid media or from agar plates were suspended in TE (10 mM Tris^ HCl, 1 mM ethylenediamine tetraacetic acid (EDTA), pH 7.5) bu¡er and adjusted to a cell density of 5U108 cells per ml. A 10-ml cell suspension was used for all RNA isolation procedures. Centrifugation was carried out at 5000Ug for 5 min at 4‡C unless noted otherwise. RNA isolation and DNase treatment by the Qiagen method were carried out as per the manufacturer’s instructions. The quantity of RNA was determined by measuring the absorbance at 260 nm, A260 (optical density (OD) 1 at A260 = 40 Wg ml31 ), using a Smartspec 3000 spectrophotometer (Bio-Rad), and its purity was determined by measuring the A260 /A280 ratio. 2.3. RNA isolation by the hot phenol extraction method A 10-ml culture was added to a tube containing 1.25 ml of ice-cold ethanol/phenol stop solution (5% water-saturated phenol, pH 6 7.0, in 95% ethanol). RNA isolation and DNase treatment were carried out as described (http://www.microarrays.org/pdfs/Total_RNA_from_Ecoli.pdf). The RNA was suspended in 50 Wl of RNase-free water and the A260 and A280 values were determined. 2.4. RNA extraction by the Triton X-100 boiling method After centrifugation, the pellet was washed with 5 ml of TE. The cells were suspended in 1 ml of TE bu¡er con-
taining 0.2% Triton X-100 (Sigma). The sample was boiled at 100‡C for 10 min and then transferred to an ice bath. An equal volume of chloroform alone or a chloroform^ methanol (1:2 or 2:1) mixture was added. The tubes were inverted 10^15 times and then centrifuged at 12 000Ug at 4‡C for 10 min. The aqueous phase was collected and treated once more with an equal volume of chloroform. After centrifugation, the RNA was then precipitated by the addition of 1/10 volume of 3 M sodium acetate, pH 5.2, 2^2.5 volumes of prechilled absolute ethanol, and incubation at 320‡C for 20 min to overnight. The precipitate was collected by centrifugation at 12 000Ug for 10 min at 4‡C. The pellet was washed twice with 1 ml of 70% ethanol and once with absolute ethanol. After centrifugation, the pellet was air-dried for 5 min and dissolved in 200 Wl of diethylpyrocarbonate (DEPC)-treated water. DNase treatment was done as described above. 2.5. RT-PCR analysis To check the suitability of RNA for RT-PCR analysis, we isolated RNA by the Triton X-100 boiling method from vancomycin-resistant E. faecium ATCC strain 51559. It was divided in three aliquots. One of the RNA aliquots was treated with DNase, the other with DNase and RNase and the third was left untreated. These RNA samples were then used to check for vanA gene-speci¢c mRNA by using vanA gene-speci¢c forward (5P-GGGAAAACGACAATTGC-3P) and reverse (5P-GTACAATGCGGCCGTTA-3P) PCR primers [11] and a one-step RT-PCR kit (Invitrogen). The reaction was carried out in 50 Wl of 1Uproprietary reaction mix containing 0.2 mM each of deoxyribonucleoside triphosphate (dNTP), 1.2 mM of MgSO4 , 0.4 WM each of the forward and reverse primers, 10 Wg of RNA, and 2.5 units of RT/platinum Taq mix. The cycling conditions were as follows: One cycle at 48‡C for 30 min (to copy cDNA from vanA genespeci¢c mRNA) followed by 94‡C denaturation for 2 min; 20 cycles at 94‡C, 15 s, 65^55‡C, 30 s (30.5‡C decrement for each cycle), 68‡C, 30 s. 15 more cycles were carried out at 94‡C, 15 s, 53‡C, 30 s, 68‡C, 30 s; and the last cycle at 68‡C, 420 s. After the cycling, samples were stored at 4‡C until electrophoresis. 2.6. Agarose gel electrophoresis The integrity of an RNA sample was tested by separating 0.2^1 Wg of RNA on a 1.2% agarose gel containing 0.66 M formaldehyde. The gels were run in MOPS electrophoresis bu¡er (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) for 3 h at 90 mV [10]. The gels were stained in ethidium bromide (1 Wg ml31 in electrophoresis bu¡er) and photographed using a gel documentation system, GDS 8000 (UVP, Inc.). PCR-ampli¢ed DNA fragment(s) were separated on a 1.5% agarose gel [12].
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Fig. 1. Comparison of RNA isolation methods. 1 Wg of RNA from E. faecium ATCC 51559 was loaded on a 1.2% agarose gel and run as described in Section 2. Lanes 1 and 7, RNA ladder; lane 2, total RNA by the Qiagen method ; lane 3, RNA by the boiling method (chloroform extraction) ; lane 4, RNA by the boiling method (chloroform^methanol (1:2) extraction); lane 5, RNA by the boiling method (chloroform^ methanol (2:1) extraction); lane 6, RNA by the hot phenol extraction method.
2.7. Northern blotting, probe labeling and hybridization For the detection of full-length transcripts of the vanA gene, 2 Wg of RNA was separated on an agarose gel and transferred to a Hybond-N membrane by vacuum blotting. The nylon membrane was subjected to hybridization with a digoxigenin (DIG)-labeled partial PCR product of the vanA gene. Probe labeling, hybridization, washing and signal detection were carried out as described [13].
3. Results and discussion Most methods for RNA isolation are time-consuming, costly, and cumbersome [1^5]. Moreover, the RNA obtained by these methods is also contaminated with s 70% of 16S and 23S rRNAs [6]. Removal of contami-
99
nating rRNA often requires an extra puri¢cation step using nucleic acid capture techniques [7^9]. The Qiagen and hot phenol extraction methods yielded RNA that was contaminated with 16S and 23S rRNA (Fig. 1, lanes 2 and 6) that needed to be removed before it could be used for molecular biology applications. However, the Triton X100 boiling method reported here provided good-quality RNA (free from contaminating 16S and 23S rRNA) (Fig. 1, lanes 3^5), contained full-length transcripts (Fig. 4) and did not require additional puri¢cation. The absence of rRNA in this preparation is probably due to heat degradation as observed earlier [16]. The quantity of RNA obtained by the boiling method ranged from 0.2 to 2 mg ml31 and had an A260 /A280 ratio between 1.9 and 2.0, which indicated a good quality of RNA (Table 1). A comparison of the A260 /A280 ratios and the yield of RNA obtained by di¡erent methods suggested comparable quality and yield (Table 1). Treatment of the boiled cells with chloroform or a chloroform^methanol mixture had no signi¢cant e¡ect on the yield or quality of RNA (Fig. 1, lanes 3^5) in triplicate experiments. RNA obtained by the Qiagen and hot phenol extraction methods was, however, contaminated with 16S and 23S rRNA (Fig. 1, lanes 2 and 6). Assuming at least 70% contamination with rRNA [6] and that 100% recovery is made in subsequent puri¢cation steps by the oligonucleotide capture method, the e¡ective yield of RNA by these methods would be only one-third of the Triton X-100 boiling method reported here. Therefore, our method is quantitatively superior to the Qiagen and hot phenol extraction methods. The e⁄cacy of this method to isolate RNA from di¡erent bacterial genera and species was demonstrated by successful isolation of RNA from ¢ve Gram-positive bacteria and four Gram-negative bacteria (Fig. 2). The quantity of RNA varied (0.2^2 mg ml31 ) depending upon the bacterial genera and species (Fig. 2, lanes 3, 8^10, and Table 1) but no di¡erences were obtained in the quality of RNA as shown by the gel patterns (Fig. 2) and A260 /A280 ratio (Table 1). A smear between 23S and 5S RNA was observed in all the RNA samples irrespective of the method
Table 1 Comparison of A260 /A280 ratios and the yield of RNA by di¡erent methods Bacteria
Gram-positive E. faecium S. aureus L. lactis L. reuteri M. vanbaalenii Gram-negative E. coli XLOLR S. typhimurium A. veronii C. jejuni
Qiagen method
Hot phenol method
A260 /A280
Yield (mg ml
2.03 2.05 1.99 1.97 1.96 2.03 1.92 2.03 2.01
)
Triton X-100 boiling method
A260 /A280
Yield (mg ml
1.02 0.92 0.65 0.83 0.22
1.99 1.98 2.01 2.03 1.96
2.40 0.20 2.40 0.23
1.99 1.96 2.01 2.01
31
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A260 /A280
Yield (mg ml31 )
0.95 1.02 0.69 0.92 0.22
2.01 2.03 2.05 1.98 1.98
1.01 0.95 0.72 0.88 0.25
2.32 0.22 2.12 0.22
1.99 1.97 2.02 1.99
2.10 0.22 1.99 0.26
31
)
100
K. Sung et al. / FEMS Microbiology Letters 229 (2003) 97^101
used for RNA isolation (Fig. 1, lanes 2^6). It is di⁄cult to tell whether it represents the degraded RNA or di¡erent size mRNA species or both. RNA preparations from E. faecium and L. reuteri also show some bands between 23S and 5S RNA (Fig. 2, lanes 2 and 5). These bands were not observed in other bacterial species (Fig. 2). At least three repetitions of the same experiment (Fig. 2) gave similar results. Since all the RNA preparations were treated with DNase, the possibility that the DNA might be contributing to the bands observed in RNA samples from E. faecium and L. reuteri was ruled out. Moreover, the presence of a 732-bp partially ampli¢ed RT-PCR product of the vanA gene from DNase-treated RNA sample of vancomycin-resistant E. faecium ATCC 51559 (Fig. 3, lane 3) also demonstrated the absence of DNA contami-
nation. However, it can also be contemplated that an incomplete removal of DNA from DNase-digested RNA could be the source of ampli¢cation. This was further disregarded by the observation that no ampli¢cation product was observed after gel analysis when DNase-treated RNA sample was further treated with RNase and used as a template in an RT-PCR reaction (Fig. 3, lane 4). Partial ampli¢cation of the vanA gene from RNA (Fig. 3, lanes 2 and 3) clearly indicated vanA gene-speci¢c mRNA (Fig. 3, lane 3). Ampli¢cation was also observed when the RNA isolated from E. faecium by hot phenol and Qiagen RNA isolation methods was used in an RT-PCR reaction (data not shown). Although, partial ampli¢cation of the vanA gene was observed from RNA irrespective of the isolation method, it did not demonstrate the integrity of the full-length transcript. To detect a full-length transcript of vanA gene-speci¢c mRNA (1031 bp), Northern-blotted RNA from E. faecium was probed with a DIG-labeled vanA PCR probe. A speci¢c hybridization signal in the region where a full-length transcript of the vanA gene would be present (Fig. 4B, lane 2) was observed, suggesting that the integrity of the RNA sample was maintained by use of this method. Boiling has been shown to degrade the rRNA [16], but no information was provided about the fate of mRNA. This study clearly demonstrates that the mRNA is protected. This is also supported by a previous study [17] where environmental samples of bacteria were collected on ¢lters and RNA was isolated by boiling in the presence of SDS^DEPC followed by acid-guanidinium extraction. The integrity of RNA was demonstrated by the ampli¢cation of various full-length naphthalene-degrading genes using RT-PCR. The Triton X-100 boiling method does not involve elaborate RNA isolation procedures, is rapid, and quantitatively yields more RNA than other methods reported here. The simplicity of the procedure, its usefulness for the isolation of RNA from Gram-
Fig. 3. Ampli¢cation of the partial vanA gene by RT-PCR. Lanes 1 and 5, 100-bp DNA ladder; lane 2, RT-PCR from DNase-untreated sample; lane 3, RT-PCR from DNase-treated RNA sample; and lane 4, RTPCR from DNase- and RNase-treated RNA sample.
Fig. 4. Northern blot analysis of full-length vanA gene transcript. Lanes 1 and 3, RNA ladder ; lane 2, RNA from E. faecium ATCC 51559 (2 Wg). A: Ethidium bromide-stained gel. B: Northern blot of gel in A probed with a DIG-labeled partial vanA gene PCR product.
Fig. 2. RNA isolation from di¡erent bacteria by the boiling chloroform extraction method. The amount of RNA loaded in each lane is indicated in parentheses after the bacterial species. Lanes 1 and 11, RNA ladder; lane 2, E. faecium (0.5 Wg); lane 3, S. aureus (0.5 Wg); lane 4, L. reuteri (1 Wg); lane 5, L. lactis (1 Wg); lane 6, A. veronii (1 Wg); lane 7, E. coli (1 Wg); lane 8, C. jejuni (0.2 Wg); lane 9, S. typhimurium (0.2 Wg); and lane 10, M. vanbaalenii (0.2 Wg).
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positive and Gram-negative bacteria, the absence of contaminating 16S and 23S rRNA, and the presence of fulllength transcripts make the Triton X-100 boiling method more desirable than other methods.
[7]
[8]
Acknowledgements The work was supported in part by the postgraduate research program at the National Center for Toxicological Research (NCTR) administered by the Oak Ridge Institute for Science and Education through an inter-agency agreement between the U.S. Department of Energy and U.S. Food and Drug Administration. We thank Drs. John Sutherland and Doug Wagner at NCTR for their critical evaluation of the manuscript.
[9]
[10]
[11]
[12]
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