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Octadecylsilyl silica column method for extraction of messenger RNA
Analytical Biochemistry 391 (2009) 72–73
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Octadecylsilyl silica column method for extraction of messenger RNA Taro Kimura a,*, Kazuo Sakurai b, Seiji Shinkai c a b c
Fukuoka Industrial Technology Center, Biotechnology and Food Research Institute, Kurume, Fukuoka 839-0861, Japan Department of Chemical Processes and Environments, University of Kitakyushu, Wakamatsu, Kitakyushu 808-0135, Japan Institute of Systems, Information Technologies, and Nanotechnologies, Sawara, Fukuoka 814-0001, Japan
a r t i c l e
i n f o
Article history: Received 13 March 2009 Available online 23 April 2009
a b s t r a c t We have developed a simple and rapid method for the purification of poly(A) tail–messenger RNA (mRNA) from total RNA by using a solid phase extraction column filled with a small amount of octadecylsilyl silica. The method is based on a hydrophobic interaction between the poly(A) tail and the octadecyl unit on the silica particle in a water/dimethyl sulfoxide mixed solution. By using this column, mRNA can be separated from 100 lg of total RNA in less than 10 min with high yields (>80%). Ó 2009 Elsevier Inc. All rights reserved.
Messenger RNA (mRNA)1 is a key element in recombinant DNA techniques and gene engineering because it directly codes for proteins. Oligo(dT)-appended devices (cellulose column, magnetic particle, and latex resin) are currently used for separating mRNA from total RNA [1,2]. This method, based on complementary hybridization between oligo(dT) and the poly(A) tail at the 30 terminal of eukaryotic mRNA, can provide highly pure mRNAs. However, the protocol is complicated and time-consuming. Therefore, there has been a persistent demand for simple and quick extraction of pure mRNA in scientific and industrial fields. As an alternative, the recognition and separation of mRNA not dependent on nucleotide/nucleotide hybridization have been investigated. For example, we demonstrated that a schizophyllan-appended column can separate mRNA from total RNA [3]. This separation is due to hydrogen bonding and hydrophobic interactions between polysaccharide and the poly(A) tail [3–6]. By around 1972, it had also been found that hydrophobic nitrocellulose membrane filters [7] and lignin-contaminated cellulose [8,9] are attracted to polypurine sequences [poly(A) tail–mRNA, poly(A), and poly(I)]. In this article, a novel mRNA extraction method using a solid phase extraction column filled with octadecylsilyl (ODS) silica (C18 column) is reported. This is a simple and rapid method for extracting mRNA from total RNA through hydrophobic interactions. ODS silica (30 mg) was packed into a plastic column with 6 mm in diameter and 55 mm in length (C18 column). ODS silica was kindly supplied by Fuji Silysia Chemical. The C18 column was washed with methanol (1.0 ml), followed by Tris–HCl buffer (100 mM, pH 7.4, 1.0 ml), before use. The experiment on RNA
* Corresponding author. Fax: +81 942 30 7244. E-mail address: taro@fitc.pref.fukuoka.jp (T. Kimura). 1 Abbreviations used: mRNA, messenger RNA; ODS, octadecylsilyl; DMSO, dimethyl sulfoxide; rRNA, ribosomal RNA; PCR, polymerase chain reaction. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.04.025
adsorption into the C18 column is described as follows. The RNA solution was prepared by mixing RNA, Tris–HCl buffer (pH 7.4), RNase free water, and variable amounts of dimethyl sulfoxide (DMSO). The RNA solution (200 ll) was added into the column and passed through with positive pressure. The eluate is denoted by fraction 1 in Fig. 2. Next, the column was washed with Tris– HCl buffer (100 mM, pH 7.4) (500 ll 2, denoted by fractions 2 and 3) and then the RNA was eluted with a methanol/water (1:1) solution (200 ll 2, denoted by fractions 4 and 5). All procedures were carried out at 5 °C. Fig. 1 compares the adsorption behavior among homo-RNAs [poly(A), poly(G), and poly(C)] in the DMSO/water mixture. The amount of RNA in every fraction was evaluated by Northern dot blot analysis. Oligo(dT)18, oligo(dC)18, and oligo(dG)18 were labeled with fluorescein–dUTP at the 30 tail by using a 30 oligo-labeling kit (Amersham Biosciences) and were used as corresponding probes for the analysis. The detection was carried out using an anti-fluorescein–alkaline phosphatase conjugate/ECF fluorescent substrate system (GE Healthcare) on a Typhoon 9200 phosphorimager (Molecular Dynamics, Amersham Pharmacia Biotech). In the presence of 0 to 50 vol% DMSO, poly(A) and poly(G) were retained in the C18 column quantitatively and eluted with more than 60 to 70 vol% DMSO. However, poly(C) was eluted with more than 30 vol% DMSO. This obvious difference owes to the ring structure of the nucleic base; that is, because polypurine is attracted strongly to hydrophobic materials, poly(A) and poly(G) are retained in the column. The adsorption behavior of mRNA and ribosomal RNA (rRNA) is shown with a dotted line in Fig. 1. mRNA sample used was separated from yeast total RNA by using an oligo(dT)/magnetic beads system (PolyATract mRNA Isolation System, Promega) according to the supplier’s instructions, and the remaining RNA mixture was used as the rRNA sample. The amount of the RNA was evaluated by Northern dot blot analysis with oligo DNA probes
73
80 60 40 20 0
0
20
40 [DMSO] (vol%)
60
80
Fig. 1. DMSO dependence of adsorption behavior of poly(A) (N), poly(G) (), poly(C) (d), mRNA (D), and rRNA (s) in the C18 column. The applied sample is 200 ll of [poly(A), poly(C), or poly(G)] = 2.5 104 lg/ll or [rRNA or mRNA] = 1.7 102 lg/ll, with [Tris–HCl (pH 7.4)] = 100 mM and [DMSO] = 0 to 70 vol%. All procedures were done at 5 °C.
corresponding to the poly(A) tail and rRNA sequence. rRNA was retained in the column in the presence of less than 30 vol% DMSO and eluted with more than 50 vol%. This feature indicates that rRNA, which consists of heteronucleotides, takes a middle behavior between polypurine and polypyrimidine. On the other hand, mRNA was retained in the column in the 0 to 50 vol% DMSO solution. This behavior is consistent with poly(A) shown in Fig. 1, indicating that the 30 terminal poly(A) tail of mRNA acts as a hydrophobic polypurine segment and interacts with the octadecyl group on the silica support. The results suggest that poly(A)–mRNA can be separated from rRNA in the presence of 30 to 50 vol% DMSO. Therefore, we tried to extract mRNA from total RNA using the poly(A)/octadecyl group interaction. Total RNA used in this report was extracted from yeast (Saccharomyces cerevisiae Kyokai no. 7). A total RNA solution (total RNA = 100 lg, [DMSO] = 45 vol%, [Tris–HCl (pH 7.4)] = 100 mM, 200 ll) was prepared and applied to the C18 column at 5 °C. The washing and eluting processes were carried out according to the above-mentioned procedure. Fig. 2A compares the distribution of the total RNA contained in fractions 1 to 5 that was estimated by ultraviolet (UV) absorbance at 260 nm analyzed on a DU7400 UV–visible spectrophotometer (Beckman). Only 5% of the applied total RNA was contained in fraction 4, and 94% of the RNA was contained in fractions 1 to 3. Next, we measured only mRNA by Northern blot analysis with the oligo(dT)18 probe. Fig. 2B compares the distribution of mRNA contained in each fraction. The results indicate that mRNA barely exists in fractions 1 to 3 (which contain most of the total RNA), whereas fraction 4 contains 80% of the mRNA. This means that a lot of poly(A)-lacking RNAs were quickly eluted in the presence of 45 vol% DMSO, whereas poly(A)–mRNA was retained in the column quantitatively. The mRNA retained in the C18 column was then recovered in fraction 4 by adding the water/methanol mixture. Therefore, this method can be used for effective and rapid extraction of mRNA from total RNA. The mRNA solutions prepared by our method and the PolyATract mRNA Isolation System were analyzed with reverse transcription real-time polymerase chain reaction (PCR) using PowerSYBR Green PCR Master Mix, MultiScribe reverse transcriptase (Applied Biosystems), and primers (GSY1: sense 50 -TTAATGAG GTCACCGCATCC-30 ; antisense 50 -CGCCAATACCCTTTTCTTCAG-30 ) on an ABI Prism 7300 Real-Time PCR System (Applied Biosystems). There was hardly any difference in mean amplification and threshold cycle (Ct) values between our method (16.78 ± 0.16) and the oligo(dT) method (16.77 ± 0.29). The C18 column method was applied to total RNA samples (50 lg) of soybean (BioChain) and rat kidney (Origene Technolo-
B
total RNA distribution (%)
A
100
mRNA distribution (%)
adsorption rate (%)
Notes & Tips / T. Kimura et al. / Anal. Biochem. 391 (2009) 72–73
80 60 40 20 0
80 60 40 20 0 1
2
3 fraction number
4
5
Fig. 2. Distribution of total RNA (A) and mRNA (B) in fractions 1 to 5 when 200 ll of the total RNA solution ([total RNA] = 0.50 lg/ll, [Tris–HCl (pH 7.4)] = 100 mM, [DMSO] = 45 vol%) was added into the C18 column. All procedures were done at 5 °C.
gies) origin, and mRNA was successfully extracted with high yields (85.1% and 90.0%, respectively). In conclusion, we have demonstrated that mRNA can be extracted in less than 10 min by using the C18 column. This method is based not on polynucleotide/polynucleotide interactions but rather on hydrophobic interactions between the poly(A) tail and octadecyl units. We anticipate that this convenient and reliable method will be a useful tool for gene engineering. Acknowledgments This work was financially supported by the Japan Science and Technology Agency’s (JST) Solution-Oriented Research for Science and Technology (SORST) program and Grant-in-Aid for Scientific Research (18750160). References [1] H. Aviv, P. Leder, Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid–cellulose, Proc. Natl. Acad. Sci. USA 69 (1972) 1408–1412. [2] C.H. Faust Jr., H. Diggelmann, B. Mach, Isolation of poly(adenylic acid)-rich ribonucleic acid from mouse myeloma and synthesis of complementary deoxyribonucleic acid, Biochemistry 12 (1973) 925–931. [3] T. Kimura, A. Beppu, K. Sakurai, S. Shinkai, Separation technique for messenger RNAs by use of schizophyllan/poly(A) tail complexation, Biomacromolecules 6 (2005) 174–179. [4] K. Sakurai, S. Shinkai, Molecular recognition of adenine, cytosine, and uracil in a single-stranded RNA by a natural polysaccharide: schizophyllan, J. Am. Chem. Soc. 122 (2000) 4520–4521. [5] K. Sakurai, M. Mizu, S. Shinkai, Polysaccharide–polynucleotide complexes: II. Complementary polynucleotide mimic behavior of the natural polysaccharide schizophyllan in the macromolecular complex with single stranded RNA and DNA, Biomacromolecules 2 (2001) 641–650. [6] T. Kimura, K. Koumoto, M. Mizu, K. Sakurai, S. Shinkai, Polysaccharide– polynucleotide interaction: XI. Novel separation system of RNAs by using schizophyllan-appended column, Chem. Lett. 12 (2002) 1240–1241. [7] G. Brawerman, J. Mendecki, S.Y. Lee, A procedure for the isolation of mammalian messenger ribonucleic acid, Biochemistry 11 (1972) 637–641. [8] P.A. Kitos, G. Saxon, H. Amos, The isolation of polyadenylate with unreacted cellulose, Biochem. Biophys. Res. Commun. 47 (1972) 1426–1437. [9] J. DeLarco, G. Guroff, The binding of RNA to various celluloses, Biochem. Biophys. Res. Commun. 50 (1973) 486–492.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Octadecylsilyl silica column method for extraction of messenger RNA Taro Kimura a,*, Kazuo Sakurai b, Seiji Shinkai c a b c
Fukuoka Industrial Technology Center, Biotechnology and Food Research Institute, Kurume, Fukuoka 839-0861, Japan Department of Chemical Processes and Environments, University of Kitakyushu, Wakamatsu, Kitakyushu 808-0135, Japan Institute of Systems, Information Technologies, and Nanotechnologies, Sawara, Fukuoka 814-0001, Japan
a r t i c l e
i n f o
Article history: Received 13 March 2009 Available online 23 April 2009
a b s t r a c t We have developed a simple and rapid method for the purification of poly(A) tail–messenger RNA (mRNA) from total RNA by using a solid phase extraction column filled with a small amount of octadecylsilyl silica. The method is based on a hydrophobic interaction between the poly(A) tail and the octadecyl unit on the silica particle in a water/dimethyl sulfoxide mixed solution. By using this column, mRNA can be separated from 100 lg of total RNA in less than 10 min with high yields (>80%). Ó 2009 Elsevier Inc. All rights reserved.
Messenger RNA (mRNA)1 is a key element in recombinant DNA techniques and gene engineering because it directly codes for proteins. Oligo(dT)-appended devices (cellulose column, magnetic particle, and latex resin) are currently used for separating mRNA from total RNA [1,2]. This method, based on complementary hybridization between oligo(dT) and the poly(A) tail at the 30 terminal of eukaryotic mRNA, can provide highly pure mRNAs. However, the protocol is complicated and time-consuming. Therefore, there has been a persistent demand for simple and quick extraction of pure mRNA in scientific and industrial fields. As an alternative, the recognition and separation of mRNA not dependent on nucleotide/nucleotide hybridization have been investigated. For example, we demonstrated that a schizophyllan-appended column can separate mRNA from total RNA [3]. This separation is due to hydrogen bonding and hydrophobic interactions between polysaccharide and the poly(A) tail [3–6]. By around 1972, it had also been found that hydrophobic nitrocellulose membrane filters [7] and lignin-contaminated cellulose [8,9] are attracted to polypurine sequences [poly(A) tail–mRNA, poly(A), and poly(I)]. In this article, a novel mRNA extraction method using a solid phase extraction column filled with octadecylsilyl (ODS) silica (C18 column) is reported. This is a simple and rapid method for extracting mRNA from total RNA through hydrophobic interactions. ODS silica (30 mg) was packed into a plastic column with 6 mm in diameter and 55 mm in length (C18 column). ODS silica was kindly supplied by Fuji Silysia Chemical. The C18 column was washed with methanol (1.0 ml), followed by Tris–HCl buffer (100 mM, pH 7.4, 1.0 ml), before use. The experiment on RNA
* Corresponding author. Fax: +81 942 30 7244. E-mail address: taro@fitc.pref.fukuoka.jp (T. Kimura). 1 Abbreviations used: mRNA, messenger RNA; ODS, octadecylsilyl; DMSO, dimethyl sulfoxide; rRNA, ribosomal RNA; PCR, polymerase chain reaction. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.04.025
adsorption into the C18 column is described as follows. The RNA solution was prepared by mixing RNA, Tris–HCl buffer (pH 7.4), RNase free water, and variable amounts of dimethyl sulfoxide (DMSO). The RNA solution (200 ll) was added into the column and passed through with positive pressure. The eluate is denoted by fraction 1 in Fig. 2. Next, the column was washed with Tris– HCl buffer (100 mM, pH 7.4) (500 ll 2, denoted by fractions 2 and 3) and then the RNA was eluted with a methanol/water (1:1) solution (200 ll 2, denoted by fractions 4 and 5). All procedures were carried out at 5 °C. Fig. 1 compares the adsorption behavior among homo-RNAs [poly(A), poly(G), and poly(C)] in the DMSO/water mixture. The amount of RNA in every fraction was evaluated by Northern dot blot analysis. Oligo(dT)18, oligo(dC)18, and oligo(dG)18 were labeled with fluorescein–dUTP at the 30 tail by using a 30 oligo-labeling kit (Amersham Biosciences) and were used as corresponding probes for the analysis. The detection was carried out using an anti-fluorescein–alkaline phosphatase conjugate/ECF fluorescent substrate system (GE Healthcare) on a Typhoon 9200 phosphorimager (Molecular Dynamics, Amersham Pharmacia Biotech). In the presence of 0 to 50 vol% DMSO, poly(A) and poly(G) were retained in the C18 column quantitatively and eluted with more than 60 to 70 vol% DMSO. However, poly(C) was eluted with more than 30 vol% DMSO. This obvious difference owes to the ring structure of the nucleic base; that is, because polypurine is attracted strongly to hydrophobic materials, poly(A) and poly(G) are retained in the column. The adsorption behavior of mRNA and ribosomal RNA (rRNA) is shown with a dotted line in Fig. 1. mRNA sample used was separated from yeast total RNA by using an oligo(dT)/magnetic beads system (PolyATract mRNA Isolation System, Promega) according to the supplier’s instructions, and the remaining RNA mixture was used as the rRNA sample. The amount of the RNA was evaluated by Northern dot blot analysis with oligo DNA probes
73
80 60 40 20 0
0
20
40 [DMSO] (vol%)
60
80
Fig. 1. DMSO dependence of adsorption behavior of poly(A) (N), poly(G) (), poly(C) (d), mRNA (D), and rRNA (s) in the C18 column. The applied sample is 200 ll of [poly(A), poly(C), or poly(G)] = 2.5 104 lg/ll or [rRNA or mRNA] = 1.7 102 lg/ll, with [Tris–HCl (pH 7.4)] = 100 mM and [DMSO] = 0 to 70 vol%. All procedures were done at 5 °C.
corresponding to the poly(A) tail and rRNA sequence. rRNA was retained in the column in the presence of less than 30 vol% DMSO and eluted with more than 50 vol%. This feature indicates that rRNA, which consists of heteronucleotides, takes a middle behavior between polypurine and polypyrimidine. On the other hand, mRNA was retained in the column in the 0 to 50 vol% DMSO solution. This behavior is consistent with poly(A) shown in Fig. 1, indicating that the 30 terminal poly(A) tail of mRNA acts as a hydrophobic polypurine segment and interacts with the octadecyl group on the silica support. The results suggest that poly(A)–mRNA can be separated from rRNA in the presence of 30 to 50 vol% DMSO. Therefore, we tried to extract mRNA from total RNA using the poly(A)/octadecyl group interaction. Total RNA used in this report was extracted from yeast (Saccharomyces cerevisiae Kyokai no. 7). A total RNA solution (total RNA = 100 lg, [DMSO] = 45 vol%, [Tris–HCl (pH 7.4)] = 100 mM, 200 ll) was prepared and applied to the C18 column at 5 °C. The washing and eluting processes were carried out according to the above-mentioned procedure. Fig. 2A compares the distribution of the total RNA contained in fractions 1 to 5 that was estimated by ultraviolet (UV) absorbance at 260 nm analyzed on a DU7400 UV–visible spectrophotometer (Beckman). Only 5% of the applied total RNA was contained in fraction 4, and 94% of the RNA was contained in fractions 1 to 3. Next, we measured only mRNA by Northern blot analysis with the oligo(dT)18 probe. Fig. 2B compares the distribution of mRNA contained in each fraction. The results indicate that mRNA barely exists in fractions 1 to 3 (which contain most of the total RNA), whereas fraction 4 contains 80% of the mRNA. This means that a lot of poly(A)-lacking RNAs were quickly eluted in the presence of 45 vol% DMSO, whereas poly(A)–mRNA was retained in the column quantitatively. The mRNA retained in the C18 column was then recovered in fraction 4 by adding the water/methanol mixture. Therefore, this method can be used for effective and rapid extraction of mRNA from total RNA. The mRNA solutions prepared by our method and the PolyATract mRNA Isolation System were analyzed with reverse transcription real-time polymerase chain reaction (PCR) using PowerSYBR Green PCR Master Mix, MultiScribe reverse transcriptase (Applied Biosystems), and primers (GSY1: sense 50 -TTAATGAG GTCACCGCATCC-30 ; antisense 50 -CGCCAATACCCTTTTCTTCAG-30 ) on an ABI Prism 7300 Real-Time PCR System (Applied Biosystems). There was hardly any difference in mean amplification and threshold cycle (Ct) values between our method (16.78 ± 0.16) and the oligo(dT) method (16.77 ± 0.29). The C18 column method was applied to total RNA samples (50 lg) of soybean (BioChain) and rat kidney (Origene Technolo-
B
total RNA distribution (%)
A
100
mRNA distribution (%)
adsorption rate (%)
Notes & Tips / T. Kimura et al. / Anal. Biochem. 391 (2009) 72–73
80 60 40 20 0
80 60 40 20 0 1
2
3 fraction number
4
5
Fig. 2. Distribution of total RNA (A) and mRNA (B) in fractions 1 to 5 when 200 ll of the total RNA solution ([total RNA] = 0.50 lg/ll, [Tris–HCl (pH 7.4)] = 100 mM, [DMSO] = 45 vol%) was added into the C18 column. All procedures were done at 5 °C.
gies) origin, and mRNA was successfully extracted with high yields (85.1% and 90.0%, respectively). In conclusion, we have demonstrated that mRNA can be extracted in less than 10 min by using the C18 column. This method is based not on polynucleotide/polynucleotide interactions but rather on hydrophobic interactions between the poly(A) tail and octadecyl units. We anticipate that this convenient and reliable method will be a useful tool for gene engineering. Acknowledgments This work was financially supported by the Japan Science and Technology Agency’s (JST) Solution-Oriented Research for Science and Technology (SORST) program and Grant-in-Aid for Scientific Research (18750160). References [1] H. Aviv, P. Leder, Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid–cellulose, Proc. Natl. Acad. Sci. USA 69 (1972) 1408–1412. [2] C.H. Faust Jr., H. Diggelmann, B. Mach, Isolation of poly(adenylic acid)-rich ribonucleic acid from mouse myeloma and synthesis of complementary deoxyribonucleic acid, Biochemistry 12 (1973) 925–931. [3] T. Kimura, A. Beppu, K. Sakurai, S. Shinkai, Separation technique for messenger RNAs by use of schizophyllan/poly(A) tail complexation, Biomacromolecules 6 (2005) 174–179. [4] K. Sakurai, S. Shinkai, Molecular recognition of adenine, cytosine, and uracil in a single-stranded RNA by a natural polysaccharide: schizophyllan, J. Am. Chem. Soc. 122 (2000) 4520–4521. [5] K. Sakurai, M. Mizu, S. Shinkai, Polysaccharide–polynucleotide complexes: II. Complementary polynucleotide mimic behavior of the natural polysaccharide schizophyllan in the macromolecular complex with single stranded RNA and DNA, Biomacromolecules 2 (2001) 641–650. [6] T. Kimura, K. Koumoto, M. Mizu, K. Sakurai, S. Shinkai, Polysaccharide– polynucleotide interaction: XI. Novel separation system of RNAs by using schizophyllan-appended column, Chem. Lett. 12 (2002) 1240–1241. [7] G. Brawerman, J. Mendecki, S.Y. Lee, A procedure for the isolation of mammalian messenger ribonucleic acid, Biochemistry 11 (1972) 637–641. [8] P.A. Kitos, G. Saxon, H. Amos, The isolation of polyadenylate with unreacted cellulose, Biochem. Biophys. Res. Commun. 47 (1972) 1426–1437. [9] J. DeLarco, G. Guroff, The binding of RNA to various celluloses, Biochem. Biophys. Res. Commun. 50 (1973) 486–492.